Antibodies specific for sclerostin and methods for increasing bone mineralization

ABSTRACT

Compositions and methods relating to antibodies that specifically bind to TGF-beta binding proteins are provided. These methods and compositions relate to altering bone mineral density by interfering with the interaction between a TGF-beta binding protein sclerostin and a TGF-beta superfamily member, particularly a bone morphogenic protein. Increasing bone mineral density has uses in diseases and conditions in which low bone mineral density typifies the condition, such as osteopenia, osteoporosis, and bone fractures.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application Ser.No. 60/478,977 filed Jun. 16, 2003.

TECHNICAL FIELD

The present invention relates generally to pharmaceutical products andmethods and, more specifically, to methods and sclerostin-specificantibodies suitable for increasing the mineral content of bone. Suchcompositions and methods may be used to treat a wide variety ofconditions including, for example, osteopenia, osteoporosis, fractures,and other disorders in which low bone mineral density is a hallmark ofthe disease.

BACKGROUND OF THE INVENTION

Two or three distinct phases of changes to bone mass occur over the lifeof an individual (see Riggs, West J. Med. 154:63-77 (1991)). The firstphase occurs in both men and women and proceeds to attainment of a peakbone mass. This first phase is achieved through linear growth of theendochondral growth plates and radial growth due to a rate of periostealapposition. The second phase begins around age 30 for trabecular bone(flat bones such as the vertebrae and pelvis) and about age 40 forcortical bone (e.g., long bones found in the limbs) and continues to oldage. This phase is characterized by slow bone loss and occurs in bothmen and women. In women, a third phase of bone loss also occurs, mostlikely due to postmenopausal estrogen deficiencies. During this phasealone, women may lose an additional 10% of bone mass from the corticalbone and 25% from the trabecular compartment (see Riggs, supra).

Loss of bone mineral content can be caused by a wide variety ofconditions and may result in significant medical problems. For example,osteoporosis is a debilitating disease in humans and is characterized bymarked decreases in skeletal bone mass and mineral density, structuraldeterioration of bone, including degradation of bone microarchitectureand corresponding increases in bone fragility, and susceptibility tofracture in afflicted individuals. Osteoporosis in humans is preceded byclinical osteopenia (bone mineral density that is greater than onestandard deviation but less than 2.5 standard deviations below the meanvalue for young adult bone), a condition found in approximately 25million people in the United States. Another 7-8 million patients in theUnited States have been diagnosed with clinical osteoporosis (defined asbone mineral content greater than 2.5 standard deviations below that ofmature young adult bone). Osteoporosis is one of the most expensivediseases for the health care system, costing tens of billions of dollarsannually in the United States. In addition to health care-related costs,long-term residential care, and lost working days add to the financialand social costs of this disease. Worldwide, approximately 75 millionpeople are at risk for osteoporosis.

The frequency of osteoporosis in the human population increases withage. Among Caucasians, osteporosis is predominant in women who, in theUnited States, comprise 80% of the osteoporosis patient pool. Theincreased fragility and susceptibility to fracture of skeletal bone inthe aged is aggravated by the greater risk of accidental falls in thispopulation. More than 1.5 million osteoporosis-related bone fracturesare reported in the United States each year. Fractured hips, wrists, andvertebrae are among the most common injuries associated withosteoporosis. Hip fractures in particular are extremely uncomfortableand expensive for the patient, and for women, correlate with high ratesof mortality and morbidity.

Although osteoporosis has been regarded as an increase in the risk offracture due to decreased bone mass, none of the presently availabletreatments for skeletal disorders can substantially increase the bonedensity of adults. A strong perception among many physicians is thatdrugs are needed that could increase bone density in adults,particularly in the bones of the wrist, spinal column, and hip that areat risk in osteopenia and osteoporosis.

Current strategies for the prevention of osteoporosis may offer somebenefit to individuals but cannot ensure resolution of the disease.These strategies include moderating physical activity (particularly inweight-bearing activities) with the onset of advanced age, providingadequate calcium in the diet, and avoiding consumption of productscontaining alcohol or tobacco. For patients presenting clinicalosteopenia or osteoporosis, the prevalent current therapeutic drugs andstrategies are directed to reducing further loss of bone mass byinhibiting the process of bone absorption, a natural aspect of the boneremodeling process that occurs constitutively.

For example, estrogen is now being prescribed to retard bone loss.However, some controversy exists over whether patients gain anylong-term benefit and whether estrogen has any effect at all on patientsover 75 years old. Moreover, use of estrogen is believed to increase therisk of breast and endometrial cancer. Calcitonin, osteocalcin withvitamin K, or high doses of dietary calcium, with or without vitamin D,have also been suggested for postmenopausal women. High doses ofcalcium, however, can often have unpleasant gastrointestinal sideeffects, and serum and urinary calcium levels must be continuouslymonitored (e.g., Khosla and Rigss, Mayo Clin. Proc. 70:978-982, 1995).

Other therapeutic approaches to osteoporosis include bisphosphonates(e.g., Fosamax™, Actonel™, Bonviva™, Zometa™, olpadronate, neridronate,skelid, bonefos), parathyroid hormone, calcilytics, calcimimetics (e.g.,cinacalcet), statins, anabolic steroids, lanthanum and strontium salts,and sodium fluoride. Such therapeutics, however, are often associatedwith undesirable side effects (for example, calcitonin and steroids maycause nausea and provoke an immune reaction, bisphosphonates and sodiumfluoride may inhibit repair of fractures, even though bone densityincreases modestly) that may preclude their efficacious use (see Khoslaand Rigss, supra).

No currently practiced therapeutic strategy for treating a conditionassociated with excessive or insufficient bone mineralization, such asosteoporosis or other disorders characterized by loss of bonemineralization, involves a drug that alters (i.e., increases ordecreases in a statistically significant manner) bone mass. Inparticular, no current strategy therapeutically stimulates or enhancesthe growth of new bone mass. The present invention provides compositionsand methods which can be used to increase bone mineralization, and whichtherefore may be used to treat a wide variety of conditions in which anincrease in bone mass is desirable. The present invention also offersother related advantages.

BRIEF SUMMARY OF THE INVENTION

Briefly stated, the present invention provides antibodies thatspecifically bind to a TGF-beta binding protein, sclerostin (SOST), andprovides immunogens comprising SOST peptides derived from regions ofSOST that interact with a member of the TGF-beta superfamily such as abone morphogenic protein. In one embodiment, the invention provides anisolated antibody, or an antigen-binding fragment thereof, that bindsspecifically to a SOST polypeptide, which comprises an amino acidsequence set forth in SEQ ID NO:1, 20, 58, 60, 62, or 68, wherein theantibody competitively inhibits binding of the SOST polypeptide to atleast one of (i) a bone morphogenic protein (BMP) Type I Receptorbinding site and (ii) a BMP Type II Receptor binding site, wherein theBMP Type I Receptor binding site is capable of binding to a BMP Type IReceptor polypeptide comprising an amino acid sequence set forth inGenBank Acc. Nos. NM_(—)004329 (SEQ ID NO:71); D89675 (SEQ ID NO:72);NM_(—)001203 (SEQ ID NO:73); S75359 (SEQ ID NO:74); NM_(—)030849 (SEQ IDNO:75); D38082 (SEQ ID NO:76); NP_(—)001194 (SEQ ID NO:77); BAA19765(SEQ ID NO:78); or AAB33865 (SEQ ID NO:79), and wherein the BMP Type IIReceptor binding site is capable of binding to a BMP Type II Receptorpolypeptide comprising the amino acid sequence set forth in GenBank Acc.NOs. U25110 (SEQ ID NO:80); NM_(—)033346 (SEQ ID NO:81); Z48923 (SEQ IDNO:83); CAA88759 (SEQ ID NO:84); or NM_(—)001204 (SEQ ID NO:82). In aparticular embodiment, the antibody, or the antigen-binding fragmentthereof, binds specifically to a polypeptide that comprises an aminoacid sequence set forth in SEQ ID NO:2-6, 12-15, 21, 22, 25, 26, 47, 48,49, or 50, and in other particular embodiments, the antibody bindsspecifically to a polypeptide that comprises an amino acid sequence setforth in SEQ ID NO: 5, 6, or 14.

In one embodiment, the invention provides an isolated antibody, or anantigen-binding fragment thereof, that is produced by immunizing anon-human animal with a peptide of at least 20 amino acids and no morethan 75 amino acids that comprises an amino acid sequence of SEQ IDNOs:2-19, 21-28, or 47-50, wherein the antibody binds specifically to aSOST polypeptide, which comprises an amino acid sequence set forth inSEQ ID NOs:1, 20, 58, 60, 62, or 68, and wherein the antibodycompetitively inhibits binding of the SOST polypeptide to at least oneof (i) a bone morphogenic protein (BMP) Type I Receptor binding site and(ii) a BMP Type II Receptor binding site, wherein the BMP Type IReceptor binding site is capable of binding to a BMP Type I Receptorpolypeptide (set forth in any one of GenBank sequences provided herein),and wherein the BMP Type II Receptor binding sites is capable of bindingto a BMP Type II Receptor polypeptide (set forth in any one of GenBanksequences provided herein). In certain preferred embodiments, anantibody is produced by immunizing a non-human animal with a peptide ofat least 20 amino acids and no more than 75 amino acids that comprisesan amino acid sequence of SEQ ID NO:5, 6, 10, 11, 14, or 18.

The present invention further provides an antibody, or anantigen-binding fragment thereof, that binds specifically to a SOSTpolypeptide and that impairs formation of a SOST homodimer, wherein theSOST polypeptide comprises an amino acid sequence set forth in SEQ IDNOs:1, 20, 58, 60, 62, or 68, and wherein the antibody binds to apolypeptide that comprises an amino acid sequence of SEQ ID NOs:29-31,33-36, 41-43, or 51-54.

In another embodiment, the invention provides an isolated antibody, oran antigen-binding fragment thereof, that is produced by immunizing anon-human animal with a peptide of at least 20 amino acids and no morethan 75 amino acids comprising an amino acid sequence of SEQ ID NOs:29-46, 51, 52, 53, or 54, wherein the antibody, or the antigen-bindingfragment thereof, binds specifically to a SOST polypeptide thatcomprises an amino acid sequence set forth in SEQ ID NO:1, 20, 58, 60,62, or 68, and wherein the antibody impairs formation of a SOSThomodimer.

In certain particular embodiments of the invention, the antibody is apolyclonal antibody. In other embodiments, the antibody is a monoclonalantibody, which is a mouse, human, rat, or hamster monoclonal antibody.The invention also provides a hybridoma cell or a host cell that iscapable of producing the monoclonal antibody. In other embodiments ofthe invention, the antibody is a humanized antibody or a chimericantibody. The invention further provides a host cell that produces thehumanized or chimeric antibody. In certain embodiments theantigen-binding fragment of the antibody is a F(ab′)₂, Fab′, Fab, Fd, orFv fragment. The invention also provides an antibody that is a singlechain antibody and provides a host cell that is capable of expressingthe single chain antibody. In another embodiment, the invention providesa composition comprising such antibodies and a physiologicallyacceptable carrier.

The invention provides an immunogen comprising a peptide comprising 6,7, 8, 9, 10, 11, or 12 consecutive amino acids of a SOST polypeptide,which SOST polypeptide comprises an amino acid sequence set forth in SEQID NOs:1, 20, 58, 60, 62, or 68, and wherein the peptide is capable ofeliciting in a non-human animal an antibody that binds specifically tothe SOST polypeptide and that competitively inhibits binding of the SOSTpolypeptide to at least one of (i) a bone morphogenic protein (BMP) TypeI Receptor binding site and (ii) a BMP Type II Receptor binding site,wherein the BMP Type I Receptor binding site is capable of binding to aBMP Type I Receptor polypeptide, wherein the BMP Type I Receptor bindingsite is capable of binding to a BMP Type I Receptor polypeptidecomprising an amino acid sequence set forth in GenBank Acc. Nos.NM_(—)004329 (SEQ ID NO:71); D89675 (SEQ ID NO:72); NM_(—)001203 (SEQ IDNO:73); S75359 (SEQ ID NO:74); NM_(—)030849 (SEQ ID NO:75); D38082 (SEQID NO:76); NP_(—)001194 (SEQ ID NO:77); BAA19765 (SEQ ID NO:78); orAAB33865 (SEQ ID NO:79), and wherein the BMP Type II Receptor bindingsite is capable of binding to a BMP Type II Receptor polypeptidecomprising the amino acid sequence set forth in GenBank Acc. NOs. U25110(SEQ ID NO:80); NM_(—)033346 (SEQ ID NO:81); Z48923 (SEQ ID NO:83);CAA88759 (SEQ ID NO:84); or NM_(—)001204 (SEQ ID NO:82).

In another embodiment, an immunogen comprises a peptide comprising atleast 21 consecutive amino acids and no more than 50 consecutive aminoacids of a SOST polypeptide, said SOST polypeptide comprising an aminoacid sequence set forth in SEQ ID NOs:1, 20, 58, 60, 62, or 68, whereinthe peptide is capable of eliciting in a non-human animal an antibodythat binds specifically to the SOST polypeptide and that competitivelyinhibits binding of the SOST polypeptide to at least one of (i) a bonemorphogenic protein (BMP) Type I Receptor binding site and (ii) a BMPType II Receptor binding site, wherein the BMP Type I Receptor bindingsite is capable of binding to a BMP Type I Receptor polypeptide (setforth in any one of GenBank sequences provided herein), and wherein theBMP Type II Receptor binding site is capable of binding to a BMP Type IIReceptor polypeptide (set forth in any one of GenBank sequences providedherein).

In certain embodiments the invention provides an immunogen comprising apeptide of at least 20 amino acids and no more than 75 amino acidscomprising an amino acid sequence of SEQ ID NOs: 2-19, 21-28, 47, 48,49, or 50, wherein the peptide is capable of eliciting in a non-humananimal an antibody that binds specifically to a SOST polypeptide, whichcomprises an amino acid sequence set forth in SEQ ID NO:1, 20, 58, 60,62, or 68, wherein the antibody competitively inhibits binding of theSOST polypeptide to at least one of (i) a bone morphogenic protein (BMP)Type I Receptor binding site and (ii) a BMP Type II Receptor bindingsite, wherein the BMP Type I Receptor binding site is capable of bindingto a BMP Type I Receptor polypeptide comprising an amino acid sequenceset forth in GenBank Acc. Nos. NM_(—)004329 (SEQ ID NO:71); D89675 (SEQID NO:72); NM_(—)001203 (SEQ ID NO:73); S75359 (SEQ ID NO:74);NM_(—)030849 (SEQ ID NO:75); D38082 (SEQ ID NO:76); NP_(—)001194 (SEQ IDNO:77); BAA19765 (SEQ ID NO:78); or AAB33865 (SEQ ID NO:79), and whereinthe BMP Type II Receptor binding site is capable of binding to a BMPType II Receptor polypeptide comprising the amino acid sequence setforth in GenBank Acc. NOs. U25110 (SEQ ID NO:80); NM_(—)033346 (SEQ IDNO:81); Z48923 (SEQ ID NO:83); CAA88759 (SEQ ID NO:84); or NM_(—)001204(SEQ ID NO:82). In a particular embodiment, the invention provides animmunogen comprising a peptide of at least 20 amino acids and no morethan 75 amino acids comprising an amino acid sequence of SEQ ID NO:5, 6,10, 11, 14, or 18.

The invention also provides an immunogen comprising a peptide of atleast 20 amino acids and no more than 75 amino acids comprising an aminoacid sequence of SEQ ID NOs: 29-46, 51, 52, 53, or 54, wherein thepeptide is capable of eliciting in a non-human animal an antibody thatbinds specifically to a SOST polypeptide, which comprises an amino acidsequence set forth in SEQ ID NO:1, 20, 58, 60, 62, or 68, and whereinthe antibody impairs formation of a SOST homodimer. In anotherembodiment, an immunogen comprises a peptide comprising 6, 7, 8, 9, 10,11, or 12 consecutive amino acids of a SOST polypeptide, wherein theSOST polypeptide comprises an amino acid sequence set forth in SEQ IDNO:1, 20, 58, 60, 62, or 68, and wherein the peptide is capable ofeliciting in a non-human animal an antibody that binds specifically tothe SOST polypeptide and that impairs formation of a SOST homodimer. Incertain embodiments, the invention provides an immunogen that comprisesa peptide comprising at least 21 consecutive amino acids and no morethan 50 consecutive amino acids of a SOST polypeptide, which polypeptidecomprises SEQ ID NO:1, 20, 58, 60, 62, or 68, and wherein the peptide iscapable of eliciting in a non-human animal an antibody that bindsspecifically to the SOST polypeptide and that impairs formation of aSOST homodimer.

In certain particular embodiments, the subject invention immunogens areassociated with a carrier molecule. In certain embodiments, the carriermolecule is a carrier polypeptide, and in particular embodiments, thecarrier polypeptide is keyhole limpet hemocyanin.

The present invention also provides a method for producing an antibodythat specifically binds to a SOST polypeptide, comprising immunizing anon-human animal with an immunogen comprising a peptide of 6, 7, 8, 9,10, 11, or 12 consecutive amino acids or at least 21 and no more than 50consecutive amino acids of a SOST polypeptide having the sequence setforth in SEQ ID NO:1, 20, 58, 60, 62, or 68, that is capable ofeliciting in a non-human animal an antibody that binds specifically tothe SOST polypeptide and that competitively inhibits binding of the SOSTpolypeptide to at least one of (i) a bone morphogenic protein (BMP) TypeI Receptor binding site and (ii) a BMP Type II Receptor binding site,wherein the BMP Type I Receptor binding site is capable of binding to aBMP Type I Receptor polypeptide, wherein the BMP Type I Receptor bindingsite is capable of binding to a BMP Type I Receptor polypeptidecomprising an amino acid sequence set forth in GenBank Acc. Nos.NM_(—)004329 (SEQ ID NO:71); D89675 (SEQ ID NO:72); NM_(—)001203 (SEQ IDNO:73); S75359 (SEQ ID NO:74); NM_(—)030849 (SEQ ID NO:75); D38082 (SEQID NO:76); NP_(—)001194 (SEQ ID NO:77); BAA19765 (SEQ ID NO:78); orAAB33865 (SEQ ID NO:79), and wherein the BMP Type II Receptor bindingsite is capable of binding to a BMP Type II Receptor polypeptidecomprising the amino acid sequence set forth in GenBank Acc. NOs. U25110(SEQ ID NO:80); NM_(—)033346 (SEQ ID NO:81); Z48923 (SEQ ID NO:83);CAA88759 (SEQ ID NO:84); or NM_(—)001204 (SEQ ID NO:82). In anotherembodiment, the present invention provides a method for producing anantibody that specifically binds to a SOST polypeptide, comprisingimmunizing a non-human animal with an immunogen comprising a peptide atleast 20 amino acids and no more than 75 amino acids comprising an aminoacid sequence of SEQ ID NO: 2-19, 21-28, 47, 48, 49, or 50, wherein thepeptide is capable of eliciting in a non-human animal an antibody thatbinds specifically to a SOST polypeptide, which comprises an amino acidsequence set forth in SEQ ID NO:1, 20, 58, 60, 62, or 68, wherein theantibody competitively inhibits binding of the SOST polypeptide to atleast one of (i) a bone morphogenic protein (BMP) Type I Receptorbinding site and (ii) a BMP Type II Receptor binding site, wherein theBMP Type I Receptor binding site is capable of binding to a BMP Type IReceptor polypeptide. In a particular embodiment, the immunogencomprises a peptide at least 20 amino acids and no more than 75 aminoacids comprising an amino acid sequence of SEQ ID NO:5, 6, 10, 11, 14,or 18.

In other preferred embodiments, the invention provides a method forproducing an antibody that specifically binds to a SOST polypeptide,comprising immunizing a non-human animal with an immunogen comprising apeptide of 6, 7, 8, 9, 10, 11, or 12 consecutive amino acids or at least21 and no more than 50 consecutive amino acids of a SOST polypeptidehaving the sequence set forth in SEQ ID NO:1, 20, 58, 60, 62, or 68,that is capable of eliciting in a non-human animal an antibody thatbinds specifically to the SOST polypeptide and that is capable ofimpairing formation of a SOST homodimer. In another embodiment, theinvention provides a method for producing an antibody that specificallybinds to a SOST polypeptide, comprising immunizing a non-human animalwith an immunogen comprising a peptide at least 20 amino acids and nomore than 75 amino acids comprising an amino acid sequence of SEQ ID NO:29-46, 51, 52, 53, or 54, wherein the peptide is capable of eliciting ina non-human animal an antibody that binds specifically to a SOSTpolypeptide and impairs formation of a SOST homodimer.

The present invention also provides a method for identifying an antibodythat modulates a TGF-beta signaling pathway, comprising contacting anantibody that specifically binds to a SOST polypeptide which comprisesan amino acid sequence set forth in any one of SEQ ID NOS: 1, 20, 58,60, 62, or 68 with at least one SOST peptide comprising an amino acidsequence of SEQ ID NO: 2-19, or 21-54, under conditions and for a timesufficient to permit formation of an antibody/SOST peptide complex; anddetecting a level of antibody/SOST peptide complex, and therebydetecting the presence of an antibody that modulates a TGF-betasignaling pathway. In another embodiment, the invention provides amethod for identifying an antibody that impairs binding of a BMP to aSOST polypeptide, comprising contacting (i) an antibody thatspecifically binds to a SOST polypeptide which comprises an amino acidsequence set forth in any one of SEQ ID NOS: 1, 20, 58, 60, 62, or 68with (ii) at least one SOST peptide comprising an amino acid sequence ofSEQ ID NO: 2-19, 21-28, or 47-50 under conditions and for a timesufficient to permit formation of an antibody/SOST peptide complex; anddetecting a level of antibody/SOST peptide complex, and therebydetecting the presence of an antibody that impairs binding of a BMP to aSOST polypeptide. In particular embodiments of the method foridentifying an antibody that modulates a TGF-beta signaling pathway andfor identifying an antibody that impairs binding of a BMP to a SOSTpolypeptide, the SOST peptide comprises an amino acid sequence of SEQ IDNO:5, 6, 10, 11, 14, or 18.

In another embodiment, the invention provides a method for identifyingan antibody that impairs SOST homodimer formation, comprising contacting(i) an antibody that specifically binds to a SOST polypeptide whichcomprises an amino acid sequence set forth in any one of SEQ ID NOS: 1,20, 58, 60, 62, or 68, with (ii) at least one SOST peptide comprising anamino acid sequence of SEQ ID NO: 29-46 or 51-54 under conditions andfor a time sufficient to permit formation of an antibody/SOST peptidecomplex; and detecting a level of antibody/SOST peptide complex, andthereby detecting the presence of an antibody that that impairs SOSThomodimer formation.

In another embodiment, the invention provides a method for identifyingan antibody that increases bone mineral content, comprising contacting(i) an antibody that specifically binds to a SOST polypeptide whichcomprises an amino acid sequence set forth in any one of SEQ ID NOS: 1,20, 58, 60, 62, or 68 with (ii) at least one SOST peptide comprising anamino acid sequence of SEQ ID NO: 2-19, or 21-54 under conditions andfor a time sufficient to permit formation of an antibody/SOST peptidecomplex; and detecting a level of antibody/SOST peptide complex, andthereby detecting the presence of an antibody that increases bonemineral content. In a particular embodiment, the peptide comprises anamino acid sequence of SEQ ID NO:5, 6, 10, 11, 14, or 18.

In certain particular embodiments for identifying an antibody thatmodulates a TGF-beta signaling pathway, for identifying an antibody thatimpairs binding of a BMP to a SOST polypeptide, for identifying anantibody that impairs SOST homodimer formation, or for identifying anantibody that increases bone mineral content, the antibody is present ina biological sample or the antibody is a purified antibody. In certainembodiments the purified antibody is a polyclonal antibody, a monoclonalantibody, a chimeric antibody, a humanized antibody, or anantigen-binding fragment of any of these antibodies.

These and other embodiments of the present invention will becomeapparent upon reference to the following detailed description andattached drawings. All references disclosed herein are herebyincorporated by reference in their entireties as if each wasincorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents an alignment of the region containing the characteristiccystine-knot of a SOST polypeptide and its closest homologues. Threedisulphide bonds that form the cystine-knot are illustrated as solidlines. An extra disulphide bond, shown by a dotted line, is unique tothis family, which connects two β-hairpin tips in the 3D structure. Thepolypeptides depicted are SOST: sclerostin (SEQ ID NO:95); CGHB: HumanChorionic Gonadotropin β (SEQ ID NO:96); FSHB: follicle-stimulatinghormone beta subunit (SEQ ID NO:97); TSHB: thyrotropin beta chainprecursor (SEQ ID NO:98); VWF: Von Willebrand factor (SEQ ID NO:99);MUC2: human mucin 2 precursor (SEQ ID NO:100); CER1: Cerberus 1 (Xenopuslaevis homolog) (SEQ ID NO:101); DRM: gremlin (SEQ ID NO:102); DAN: (SEQID NO:103); CTGF: connective tissue growth factor precursor (SEQ IDNO:104); NOV: NovH (nephroblastoma overexpressed gene protein homolog)(SEQ ID NO:105); CYR6: (SEQ ID NO:106).

FIG. 2 illustrates a 3D model of the core region of SOST (SOST_Core).

FIG. 3 presents a 3D model of the core region of SOST homodimer.

FIGS. 4A and B provides an amino acid sequence alignment of Noggin fromfive different animals: human (NOGG_HUMAN (SEQ ID NO:107); chicken(NOGG_CHICK, SEQ ID NO:108); African clawed frog (NOGG_XENLA, SEQ IDNO:109); NOGG_FUGRU, SEQ ID NO:110); and zebrafish (NOGG_ZEBRA, SEQ IDNO:111); and SOST from human (SOST_HUMAN, SEQ ID NO:1), rat (SOST_RAT,SEQ ID NO:20), and mouse (SOST_Mouse, SEQ ID NO:112).

FIG. 5 illustrates the Noggin/BMP-7 complex structure. The BMP homodimeris shown on the bottom portion of the figure in surface mode. The Nogginhomodimer is shown on top of the BMP dimer in cartoon mode. The circlesoutline the N-terminal binding region, the core region, and the linkerbetween the N-terminal and core regions.

FIG. 6 depicts a 3D model of the potential BMP-binding fragment locatedat the SOST N-terminal region. A BMP dimer is shown in surface mode, andthe potential BMP-binding fragment is shown in stick mode. Aphenylalanine residue fitting into a hydrophobic pocket on the BMPsurface is noted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibodies that specifically bind to aSOST polypeptide methods for using such antibodies. The presentinvention also provides SOST polypeptide immunogens that may be used forgeneration and analysis of these antibodies. The antibodies may beuseful to block or impair binding of a SOST polypeptide, which is aTGF-beta binding protein, to a ligand, particularly a bone morphogenicprotein, and may also block or impair binding of the SOST polypeptide toone or more other ligands.

The invention relates in part to the surprising discovery, within thesclerostin polypeptide sequence, of specific short peptide sequencesthat are specifically recognized by anti-sclerostin antibodies capableof competitively inhibiting binding of sclerostin polypeptides to BMP,where such sclerostin-BMP binding otherwise would occur via a BMP Type IReceptor binding site and/or a BMP Type II Receptor binding site. Amolecule such as an antibody that inhibits the binding of the TGF-betabinding protein to one or more members of the TGF-beta family ofproteins, including one or more bone morphogenic proteins (BMPs), shouldbe understood to include, for example, a molecule that allows theactivation of a TGF-beta family member or BMP, or allows binding ofTGF-beta family members including one or more BMPs to their respectivereceptors by removing or preventing the TGF-beta member from binding tothe TGF-binding-protein.

The present invention also provides peptide and polypeptide immunogensthat, unexpectedly, may be used to generate and/or identify antibodiesor fragments thereof that are capable of inhibiting, preventing, orimpairing (e.g., decreasing in a statistically significant manner)binding of the TGF-beta binding protein SOST to one or more BMPs.Exemplary peptide immunogens may comprise 6, 7, 8, 9, 10, 11, 12, 20-25,21-50, 26-30, 31-40, 41-50, 51-60, 61-70, or 71-75 consecutive aminoacids of a sclerostin polypeptide as provided herein (or of a variantthereof), such peptides comprising, for instance, amino acid sequencesas set forth in SEQ ID NOS:2-19, 21-53, and 54. The present inventionalso provides peptide and polypeptide immunogens that may be used togenerate and/or identify antibodies or fragments thereof that arecapable of inhibiting, preventing, or impairing the formation of SOSThomodimers. The antibodies of the present invention are useful forincreasing the mineral content and mineral density of bone, therebyameliorating numerous conditions that result in the loss of bone mineralcontent, including for example, disease, genetic predisposition,accidents that result in the lack of use of bone (e.g., due tofracture), therapeutics that effect bone resorption or that kill boneforming cells, and normal aging.

Sclerosteosis

Sclerosteosis is a disease related to abnormal bone mineral density inhumans. Sclerosteosis is a term that was applied by Hansen (Hansen, H.G., Sklerosteose in: Opitz et al., eds. Handbuch der Kinderheilkunde,(Berlin: Springer 1967) 351-355) to a disorder similar to van Buchemhyperostosis corticalis generalisata but possibly differing inradiologic appearance of the bone changes and in the presence ofasymmetric cutaneous syndactyly of the index and middle fingers in manycases. Sclerosteosis is now understood to be an autosomal semi-dominantdisorder that is characterized by widely disseminated sclerotic lesionsof the bone in the adult. The condition is progressive. Sclerosteosisalso has a developmental aspect that is associated with syndactyly (twoor more fingers fused together). Sclerosteosis Syndrome is associatedwith large stature, and many affected individuals attain a height of sixfeet or more. In addition, the jaw of persons with this condition has anunusually square appearance. The bone mineral content of homozygotes canbe 1 to 6 fold greater than normal individuals, and bone mineral densitycan be 1 to 4 fold above normal values (e.g., in comparison tounaffected siblings).

Sclerosteosis Syndrome occurs primarily in Afrikaaners of Dutch descentin South Africa. Approximately 1 of every 140 individuals in theAfrikaaner population is a carrier of the mutated gene (heterozygotes).The mutation shows 100% penetrance. Anecdotal reports indicate thatincreased bone mineral density has been observed in heterozygotes butthey have no associated pathologies (e.g., syndactyly or skullovergrowth).

No abnormality of the pituitary-hypothalamus axis has been observed inpatients with sclerosteosis. In particular, no over-production of growthhormone and cortisone occurs, and sex hormone levels are normal inaffected individuals. Bone turnover markers (such as osteoblast specificalkaline phosphatase, osteocalcin, type 1 procollagen C′ propeptide(PICP), and total alkaline phosphatase (see Comier, Curr. Opin. in Rheu.7:243 (1995)) indicate that hyperosteoblastic activity is associatedwith the disease but normal to slightly decreased osteoclast activity isobserved as measured by markers of bone resorption (pyridinoline,deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasmatartrate-resistant acid phosphatases, and galactosyl hydroxylysine (seeComier, supra)).

Sclerosteosis is characterized by the continual deposition of bonethroughout the skeleton during the lifetime of the affected individuals.In homozygotes the continual deposition of bone mineral leads to anovergrowth of bone in areas of the skeleton where mechanoreceptors areabsent (e.g., skull, jaw, cranium). In homozygotes with Sclerosteosis,the overgrowth of the bones of the skull leads to cranial compressionand eventually to death due to excessive hydrostatic pressure on thebrain stem. A generalized and diffuse sclerosis is observed in all otherparts of the skeleton. Cortical areas of the long bones are greatlythickened resulting in a substantial increase in bone strength.Trabecular connections are increased in thickness, which in turnincreases the strength of the trabecular bone. Sclerotic bones appearunusually opaque to x-rays.

The rare genetic mutation that is responsible for Sclerosteosis Syndromehas been localized to the region of human chromosome 17. A gene withinthis region encodes a novel member of the TGF-beta binding-proteinfamily (see, e.g., U.S. Pat. Nos. 6,395,511, 6,489,445, and 6,495,736;Brunkow et al., Am. J. Hum. Genet. 68:577-89 (2001)). Therapeuticsrelated to altering or modulating the level of sclerostin may thereforebe useful in treating conditions and diseases related to abnormal bonedevelopment or deterioration. As described in more detail below,antibodies that specifically bind to this TGF-beta binding-protein,sclerostin (also referred to herein as Beer or as SOST), may be used toincrease bone mineral content thus treating, preventing, retardingprogression, or ameliorating the symptoms of a number of diseases.

TGF-Beta Superfamily

Among significant molecules to which reference is made herein includeany known or novel members of the Transforming Growth Factor-beta(TGF-beta) superfamily, which includes bone morphogenic proteins (BMPs).Also included in the TGF-beta superfamily are TGF-beta receptors, whichshould be understood to refer to one or more receptors that are specificfor a particular member of the TGF-beta superfamily (including BMPs),and TGF-beta binding proteins, which should be understood to refer toone or more polypeptides with specific binding affinity for a particularmember or subset of members of the TGF-beta superfamily (includingBMPs). A specific example of a TGF-beta binding protein includessclerostin or SOST. Polynucleotide sequences encoding SOST and SOSTvariants in various animals, including humans, are provided herein inSEQ ID NOs: 55, 56, 57, 59, 61, 63, 65, and 67 and 69 (the encodedpolypeptide sequences are provided in SEQ ID NOs: 1, 20, 58, 60, 62, 64,66, 68, and 70, respectively). (See, e.g., U.S. Pat. Nos. 6,395,511;6,489,445; and 6,495,736. See also e.g., Balemans et al., 2002 Dev.Biol. 250:231; Schmitt et al., 1999J. Orthopaed. Res. 17:269; Khalil,1999 Microbes Infect. 1:1255; Miyazono et al., 1993 Growth Factors 8:11;von Bubnoff et al., 2001 Dev. Biol. 239:1; Koli et al., 2001 Microsc.Res. Tech. 52:354; Ebara et al., 2002 Spine 27(16 Suppl. 1):S10;Bondestam, 2002, Ligands & Signaling Components of the TransformingGrowth Factor β Family, Helsinki University Biomedical Dissertations No.17).

The TGF-beta superfamily contains a variety of growth factors that sharecommon sequence elements and structural motifs at both secondary andtertiary levels. This protein family exerts a wide spectrum ofbiological responses that affect a large variety of cell types. Many ofthe members of the TGF-beta family have important functions in patternformation and tissue specification during embryonal development. Inadults, TGF-beta family members are involved, for example, in woundhealing, bone repair and bone remodeling, and in modulation of theimmune system. In addition to the TGF-beta polypeptides, thissuperfamily includes BMPs, activins, inhibins, Growth andDifferentiation Factors (GDFs), and Glial-Derived Neurotrophic Factors(GDNFs). Primary classification is established through general sequencefeatures that bin a specific protein into a general subfamily.Additional stratification within the sub-family is possible due tostricter sequence conservation between members of the smaller group. Incertain instances, such as with BMP-5, BMP-6, and BMP-7, the amino acidpercent identity may be as high as 75% among members of the smallergroup. This level of identity enables a single representative sequenceto illustrate the key biochemical elements of the sub-group thatseparates it from other members of the larger family.

The crystal structure of TGF-beta2 has been determined. The general foldof the TGF-beta2 monomer contains a stable, compact, cysteine knotlikestructure formed by three disulphide bridges. Dimerization, which isstabilized by one disulphide bridge, is antiparallel.

TGF-beta family members signal by inducing the formation ofhetero-oligomeric receptor complexes. Transduction of TGF-beta signalsinvolves two distinct subfamilies of transmembrane serine/threoninekinase receptors, type I and type II. At least seven type I receptorsand five type II receptors have been identified (see Kawabata et al.,Cytokine Growth Factor Rev. 9:49-61 (1998); Miyazono et al., Adv.Immunol. 75:115-57 (2000). Each member of the TGF-beta family binds to acharacteristic combination of type I and type II receptors, both ofwhich are needed for signaling. In the current model for TGF-betareceptor activation, a TGF-beta ligand first binds to the type IIreceptor (TbR-II), which then recruits a type I receptor (TbR-I) to forma ligand/type II/type I ternary complex. The type I receptor, cannotbind ligand in the absence of TbR-II. TbR-II then phosphorylates TbR-Ipredominantly in a domain rich in glycine and serine residues (GSdomain) in the juxtamembrane region, thereby activating TbR-I. Theactivated type I receptor kinase then phosphorylates particular membersof the Smad family of proteins that translocate to the nucleus wherethey modulate transcription of specific genes.

Bone Morphogenic Proteins (BMPs): Key Regulatory Proteins of BoneMineral Density

A major advance in the understanding of bone formation was theidentification of the BMPs, also known as osteogenic proteins (OPs),which regulate cartilage and bone differentiation in vivo. BMPs/OPsinduce endochondral bone differentiation through a cascade of events bywhich mesenchymal stem cells differentiate into chondrocytes that laydown a cartilage structure that is reabsorbed and replaced by bonetissue (see Balemans et al., Dev. Biol. 250:231-50 (2002)). Thus, thisprocess involves formation of cartilage, hypertrophy and calcificationof the cartilage, vascular invasion, differentiation of osteoblasts, andformation of bone. As described above, the BMPs/OPs (BMP 2-14, andosteogenic protein 1 and -2, OP-1 and OP-2) (see, e.g., GenBank P12643(BMP-2); GenBank P12645 (BMP3); GenBank P55107 (BMP-3b,Growth/differentiation factor 10) (GDF-10)); GenBank P12644 (BMP4);GenBank P22003 (BMP5); GenBank P22004 (BMP6); GenBank P18075 (BMP7);GenBank P34820 (BMP8); GenBank Q9UK05 (BMP9); GenBank 095393 (BM10);GenBank 095390 (BMP11, Growth/differentiation factor 11 precursor(GDF-11)); GenBank 095972 (BM15)) are members of the TGF-betasuperfamily. The striking evolutionary conservation among members of theBMP/OP sub-family suggests that they are important in the normaldevelopment and function of animals. In addition to postfetalchondrogenesis and osteogenesis, the BMPs/OPs play multiple roles inskeletogenesis, including the development of craniofacial and dentaltissues. Different BMP family members also have biological activities invarious other cell types, including monocytes, epithelial cells,mesenchymal cells, and neuronal cells. BMPs regulate cell proliferationand differentiation, chemotaxis and apoptosis, and also controlfundamental roles, for example, left-right asymmetry, neurogenesis,mesoderm patterning, and embryonic development and organogenesis of anumber of organs including the kidney, gut, lung, teeth, limb, amnion,and testis (see Balemans, supra).

BMPs are synthesized as large precursor proteins. Upon dimerization, theBMPs are proteolyically cleaved within the cell to yieldcarboxy-terminal mature proteins that are then secreted from the cell.BMPs, like other TGF-beta family members, initiate signal transductionby binding cooperatively to both type I and type II serine/threoninekinase receptors. Type I receptors for which BMPs may act as ligandsinclude BMPR-IA (also known as ALK-3), BMPR-IB (also known as ALK-6),ALK-1, and ALK-2 (also known as ActR-I). Of the type II receptors, BMPsbind to BMP type II receptor (BMPR-II), Activin type II (ActR-II), andActivin type IIB (ActR-IIB). (See Balemans et al., supra, and referencescited therein). Polynucleotide sequences and the encoded amino acidsequence of BMP type I receptor polypeptides are provided in the GenBankdatabase, for example, GenBank NM_(—)004329 (SEQ ID NO:71 encoded by SEQID NO:85); D89675 (SEQ ID NO:72 encoded by SEQ ID NO:86); NM_(—)001203(SEQ ID NO:73 encoded by SEQ ID NO:87); S75359 (SEQ ID NO:74 encoded bySEQ ID NO:88); NM_(—)030849 (SEQ ID NO:75 encoded by SEQ ID NO:89); andD38082 (SEQ ID NO:76 encoded by SEQ ID NO:90). Other polypeptidesequences of type I receptors are provided in the GenBank database, forexample, NP_(—)001194 (SEQ ID NO:77); BAA19765 (SEQ ID NO:78); andAAB33865 (SEQ ID NO:79). Polynucleotide sequences and the encoded aminoacid sequence of BMP type II receptor polypeptides are provided in theGenBank database and include, for example, U25110 (SEQ ID NO:80 encodedby SEQ ID NO:91); NM_(—)033346 (SEQ ID NO:81 encoded by SEQ ID NO:92);NM_(—)001204 (SEQ ID NO:82 encoded by SEQ ID NO:93); and Z48923 (SEQ IDNO:83 encoded by SEQ ID NO:94). Additional polypeptide sequences of typeII receptors are also provided in the GenBank database, for example,CAA88759 (SEQ ID NO:84).

BMPs, similar to other cystine-knot proteins, form a homodimer structure(Scheufler et al., J. Mol. Biol. 287:103-15 (1999)). According toevolutionary trace analysis performed on the BMP/TGF-β family, the BMPtype I receptor binding site and type II receptor binding site weremapped to the surface of the BMP structure (Innis et al., Protein Eng.13:839-47 (2000)). The location of the type I receptor binding site onBMP was later confirmed by the x-ray structure of BMP-2/BMP Receptor IAcomplex (Nickel et al., J. Joint Surg. Am. 83A(Suppl 1(Pt 1)):S7-S14(2001)). The predicted type II receptor binding site is in goodagreement with the x-ray structure of TGF-β3/TGF-β Type II receptorcomplex (Hart et al., Nat. Struct. Biol. 9:203-208 (2002)), which ishighly similar to the BMP/BMP Receptor IIA system.

The BMP and Activin sub-families are subject to significantpost-translational regulation by TGF-beta binding proteins. An intricateextracellular control system exists, whereby a high affinity antagonistis synthesized and exported, and subsequently forms a complexselectively with BMPs or activins to disrupt their biological activity(Smith, Trends Genet. 15:3-6 (1999)). A number of such TGF-beta bindingproteins have been identified, and on the basis of sequence divergence,the antagonists appear to have evolved independently because of the lackof primary sequence conservation. In vertebrates, antagonists includenoggin, chordin, chordin-like, follistatin, FSRP, the DAN/Cerberusprotein family, and sclerostin (SOST) (see Balemans et al., supra, andreferences cited therein). The mechanism of antagonism seems to differfor the different antagonists (Iemura et al. (1998) Proc. Natl. Acad.Sci. USA 95:9337-9342).

The type I and type II receptor binding sites on the BMP antagonistnoggin have also been mapped. Noggin binds to BMPs with high affinity(Zimmerman et al., 1996). A study of the noggin/BMP-7 complex structurerevealed the binding interactions between the two proteins (Groppe etal., Nature 420:636-42 (2002)). Superposition of the noggin-BMP-7structure onto a model of the BMP signaling complex showed that nogginbinding effectively masks both pairs of binding epitopes (i.e., BMP TypeI and Type II receptor binding sites) on BMP-7. The cysteine-richscaffold sequence of noggin is preceded by an N-terminal segment ofabout 20 amino acid residues that are referred to as the “clip”(residues 28-48). The type I receptor-binding site is occluded by theN-terminal portion of the clip domain of Noggin, and the type IIreceptor binding site is occluded by the carboxy terminal portion of theclip domain. Two β-strands in the core region near the C-terminus ofnoggin also contact BMP-7 at the type II receptor binding site. Thisbinding mode enables a Noggin dimer to efficiently block all thereceptor binding sites (two type I and two type II receptor bindingsites) on a BMP dimer.

Sclerostin Polypeptides and Encoding Polynucleotides The BMP antagonistsclerostin (U.S. Pat. Nos. 67,395,511, 6,489,445, and 6,495,736; seealso SEQ ID NOs: 1, 20, 58, 60, 62, 64, 66, 68, and 70), possesses anearly identical cysteine (disulfide) scaffold when compared with HumanDAN, Human Gremlin, and Human Cerberus, and SCGF (U.S. Pat. No.5,780,263) but almost no homology at the nucleotide level (forbackground information, see generally Hsu et al., Mol. Cell. 1:673-683(1998)). Sclerostin should also be understood to include variants ofthis TGF-beta binding-protein (e.g., SEQ ID NOs. 60 and 62). As usedherein, a TGF-beta binding-protein variant polynucleotide refers tonucleic acid molecule that encodes a polypeptide having an amino acidsequence that is a modification (insertion, deletion, or substitution ofone or more nucleotides) of SEQ ID NOs: 55-57, 59, 61, 63, 65, 67, or69. Such variants include naturally-occurring polymorphisms or allelicvariants of TGF-beta binding-protein encoding polynucleotides, as wellas synthetic polynucleotides that encode conservative amino acidsubstitutions of these amino acid sequences. A variety of criteria knownto those skilled in the art indicate whether amino acids at a particularposition in a peptide or polypeptide are similar. For example, a similaramino acid or a conservative amino acid substitution is one in which anamino acid residue is replaced with an amino acid residue having asimilar side chain, which include amino acids with basic side chains(e.g., lysine, arginine, histidine); acidic side chains (e.g., asparticacid, glutamic acid); uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine,histidine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan);beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).Proline, which is considered more difficult to classify, sharesproperties with amino acids that have aliphatic side chains (e.g., Leu,Val, Ile, and Ala). In certain circumstances, substitution of glutaminefor glutamic acid or asparagine for aspartic acid may be considered asimilar substitution in that glutamine and asparagine are amidederivatives of glutamic acid and aspartic acid, respectively.

Additional variant forms of a TGF-beta binding protein encodingpolynucleotide are nucleic acid molecules that contain substitutions,insertions or deletions of one or more nucleotides found within thenucleotide sequences described herein. A variant or mutant of a TGF-betabinding protein may be constructed or identified so that the alteredversion of the TGF-beta binding protein competes with the wildtypeTGF-beta binding protein. Such competition would preferably block theactivity of the wildtype TGF-beta binding protein and thus lead toincreased bone density.

An isolated polynucleotide is a nucleic acid molecule (polynucleotide oroligonucleotide) that is not integrated in the genomic DNA of anorganism. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment. For example, a DNA molecule that encodes a TGF-bindingprotein that has been separated from the genomic DNA of a eukaryoticcell is an isolated DNA molecule. Another example of an isolated nucleicacid molecule is a chemically-synthesized nucleic acid molecule that isnot integrated into the genome of an organism. The isolated nucleic acidmolecule may be DNA, cDNA, RNA, or composed at least in part of nucleicacid analogs.

TGF-beta binding-protein variant polynucleotides can be identified bydetermining whether the polynucleotides hybridize with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NOs: 55-57, 59, 61,63, 65, 67, or 69 under stringent conditions. In addition, TGF-betabinding-protein variant polynucleotides encode a protein having acysteine backbone. TGF-beta binding-protein variant polynucleotides mayalso be identified by sequence comparison. As used herein, two aminoacid sequences have “100% amino acid sequence identity” if the aminoacid residues of the two amino acid sequences are the same when alignedfor maximal correspondence. Similarly, two nucleotide sequences have“100% nucleotide sequence identity” if the nucleotides of the twonucleotide sequences are the same when aligned for maximalcorrespondence. Sequence comparisons can be performed using standardsoftware programs such as those included in the LASERGENE bioinformaticscomputing suite, which is produced by DNASTAR (Madison, Wis.), or theBLAST algorithm available at the NCBI web site([Internet]<:http:www.ncbi.nlm.nih.gov>). Other methods for comparingtwo or more nucleotide or amino acid sequences by determining optimalalignment are well-known to those of skill in the art (see, for example,Peruski and Peruski, The Internet and the New Biology: Tools for Genomicand Molecular Research (ASM Press, Inc. 1997), Wu et al. (eds.),“Information Superhighway and Computer Databases of Nucleic Acids andProteins,” in Methods in Gene Biotechnology, pages 123-151 (CRC Press,Inc. 1997), and Bishop (ed.), Guide to Human Genome Computing, 2ndEdition (Academic Press, Inc. 1998)).

A variant TGF-beta binding protein should have at least a 50% amino acidsequence identity to SEQ ID NOs: 1, 20, 58, 60, 62, 64, 66, and 68, andpreferably greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%identity. Alternatively, TGF-beta binding-protein variants can beidentified by having at least a 70% nucleotide sequence identity to SEQID NOs: 55-57, 59, 61, 63, 65, 67, or 69. Moreover, the presentinvention contemplates TGF-beta binding-protein polynucleotide variantshaving greater than 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:55or 57. Regardless of the particular method used to identify a TGF-betabinding-protein variant polynucleotide or variant TGF-beta bindingprotein, such a variant can be functionally characterized by, forexample, its ability to bind to and/or inhibit the signaling of aselected member of the TGF-beta family of proteins, or by its ability tobind specifically to an anti-TGF-beta binding-protein antibody.

Within the context of this invention, a “functional fragment” of aTGF-beta binding-protein polynucleotide refers to a nucleic acidmolecule that encodes a portion of a TGF-beta binding-proteinpolypeptide that either (1) possesses a functional activity as describedherein or (2) specifically binds to an anti-TGF-beta binding proteinantibody. For example, a functional fragment of a TGF-beta bindingprotein encoding polynucleotide described herein comprises a portion ofthe nucleotide sequence of SEQ ID Nos: 55-57, 59, 61, 63, 65, 67, or 69.

An “isolated polypeptide” referred to herein is a polypeptide that isremoved from its natural environment. For example, a naturally-occurringprotein is isolated if it is separated from some or all of thecoexisting materials in the natural system, such as carbohydrate, lipid,nucleic acids, or proteinaceous impurities associated with thepolypeptide in nature. Preferably, such isolated polypeptides are atleast about 90% pure, more preferably at least about 95% pure, and mostpreferably at least about 99% pure.

Antibodies Specific for TGF-Beta Binding Proteins

The present invention provides antibodies that specifically bind to SOSTand also provides SOST polypeptide immunogens that may be used forgeneration and analysis of these antibodies and also provides methodsfor using such antibodies. The antibodies of the present invention asdescribed herein specifically bind to a SOST polypeptide and therebyblock or inhibit the binding of a BMP to the SOST polypeptide, that is,prevent or impair the interaction between the BMP and SOST. The effectof blocking this interaction is to alter (increase or decrease in astatistically significant manner) bone mineral density, preferably toincrease bone mineral density.

Polypeptides or peptides useful for immunization and/or analysis ofSOST-specific antibodies may also be selected by analyzing the primary,secondary, and tertiary structure of a TGF-beta binding proteinaccording to methods known to those skilled in the art and describedherein, in order to determine amino acid sequences more likely togenerate an antigenic response in a host animal. See, e.g., Novotny,Mol. Immunol. 28:201-207 (1991); Berzofsky, Science 229:932-40 (1985)).Modeling and x-ray crystallography data may also be used to predictand/or identify which portions or regions of a TGF-beta binding proteininteract with which portions of a TGF-beta binding protein ligand, suchas a BMP. TGF-beta binding protein peptide immunogens may be designedand prepared that include amino acid sequences within or surrounding theportions or regions of interaction. These antibodies may be useful toblock or impair binding of the TGF-beta binding protein to the sameligand and may also block or impair binding of the TGF-beta bindingprotein to one or more other ligands.

Antibodies or antigen binding fragments thereof contemplated by thepresent invention include antibodies that are capable of specificallybinding to SOST and competitively inhibiting binding of a TGF-betapolypeptide, such as a BMP, to SOST. For example, the antibodiescontemplated by the present invention competitively inhibit binding ofthe SOST polypeptide to the BMP Type I receptor site on a BMP, or to theBMP Type II receptor binding site, or may competitively inhibit bindingof SOST to both the Type I and Type II receptor binding sites on a BMP.Without wishing to be bound by theory, when an anti-SOST antibodycompetitively inhibits binding of the Type I and/or Type II bindingsites of the BMP polypeptide to SOST, thus blocking the antagonisticactivity of SOST, the receptor binding sites on BMP are available tobind to the Type I and Type II receptors, thereby increasing bonemineralization. The binding interaction between a TGF-beta bindingprotein such as SOST and a TGF-beta polypeptide such as a BMP generallyoccurs when each of the ligand pairs forms a homodimer. Thereforeinstead of or in addition to using an antibody specific for SOST toblock, impair, or prevent binding of SOST to a BMP by competitivelyinhibiting binding of SOST to BMP, a SOST specific antibody may be usedto block or impair SOST homodimer formation.

By way of example, one dimer of human Noggin, which is a BMP antagonistthat has the ability to bind a BMP with high affinity (Zimmerman et al.,supra), was isolated in complex with one dimer of human BMP-7 andanalyzed by multiwavelength anomalous diffraction (MAD) (Groppe et al.,Nature 420:636-42 (2002)). As discussed herein, this study revealed thatNoggin dimer may efficiently block all the receptor binding sites (twotype I and two type II receptor binding sites) on a BMP dimer. Thelocation of the amino acids of Noggin that contact BMP-7 may be usefulin modeling the interaction between other TGF-beta binding proteins,such as sclerostin (SOST), and BMPs, and thus aiding the design ofpeptides that may be used as immunogens to generate antibodies thatblock or impair such an interaction.

In one embodiment of the present invention, an antibody, or anantigen-binding fragment thereof, that binds specifically to a SOSTpolypeptide competitively inhibits binding of the SOST polypeptide to atleast one or both of a bone morphogenic protein (BMP) Type I Receptorbinding site and a BMP Type II Receptor binding site that are located ona BMP. The epitopes on SOST to which these antibodies bind may includeor be included within contiguous amino acid sequences that are locatedat the N-terminus of the SOST polypeptide (amino acids at aboutpositions 1-56 of SEQ ID NO:1). The polypeptides may also include ashort linker peptide sequence that connects the N-terminal region to thecore region, for example, polypeptides as provided in SEQ ID NO:47(human) and SEQ ID NO:48 (rat). Shorter representative N-terminuspeptide sequences of human SOST (e.g., SEQ ID NO:1) include SEQ IDNOS:2-6, and representative rat SOST (e.g., SEQ ID NO:20) peptidesequences include SEQ ID NOS:12-15.

Antibodies that specifically bind to a SOST polypeptide and block orcompetitively inhibit binding of the SOST polypeptide to a BMP, forexample, by blocking or inhibiting binding to amino acids of a BMPcorresponding to one or more of the Type I and Type II receptor bindingsites may also specifically bind to peptides that comprise an amino acidsequence corresponding to the core region of SOST (amino acids at aboutpositions 57-146 of SEQ ID NO:1). Polypeptides that include the coreregion may also include additional amino acids extending at either orboth the N-terminus and C-terminus, for example, to include cysteineresidues that may be useful for conjugating the polypeptide to a carriermolecule. Representative core polypeptides of human and rat SOST, forexample, comprise the amino acid sequences set forth in SEQ ID NO:49 andSEQ ID NO:50, respectively. Such antibodies may also bind shorterpolypeptide sequences. Representative human SOST core peptide sequencesare provided in SEQ ID NOs:21-24 and representative rat SOST coresequences are provided in SEQ ID NOs:25-28.

In another embodiment, antibodies that specifically bind to a SOSTpolypeptide impair (inhibit, prevent, or block, e.g., decrease in astatistically significant manner) formation of a SOST homodimer. Becausethe interaction between SOST and a BMP may involve a homodimer of SOSTand a homodimer of the BMP, an antibody that prevents or impairshomodimer formation of SOST may thereby alter bone mineral density,preferably increasing bone mineral density. In one embodiment,antibodies that bind to the core region of SOST prevent homodimerformation. Such antibodies may also bind to peptides that comprisecontiguous amino acid sequences corresponding the core region, forexample, SEQ ID NOs: 29, 30 and 53 (human SOST) and SEQ ID NOs:31 and 54(rat SOST). Antibodies that bind to an epitope located on the C-terminalregion of a SOST polypeptide (at about amino acid positions 147-190 ofeither SEQ ID NO:1 or 20) may also impair homodimer formation.Representative C-terminal polypeptides of human and rat SOST, forexample, comprise the amino acid sequences set forth in SEQ ID NO:51 andSEQ ID NO:52, respectively. Such antibodies may also bind shorterpolypeptide sequences. Representative human SOST C-terminal peptidesequences are provided in SEQ ID NOs:33-36 and representative rat SOSTC-terminal sequences are provided in SEQ ID NOs:41-43.

The SOST polypeptides and peptides disclosed herein to which antibodiesmay specifically bind are useful as immunogens. These immunogens of thepresent invention may be used for immunizing an animal to generate ahumoral immune response that results in production of antibodies thatspecifically bind to a Type I or Type II receptor binding site or bothlocated on a BMP include peptides derived from the N-terminal region ofSOST or that may prevent SOST homodimer formation.

Such SOST polypeptides and peptides that are useful as immunogens mayalso be used in methods for screening samples containing antibodies, forexample, samples of purified antibodies, antisera, or cell culturesupernatants or any other biological sample that may contain one or moreantibodies specific for SOST. These peptides may also be used in methodsfor identifying and selecting from a biological sample one or more Bcells that are producing an antibody that specifically binds to SOST(e.g., plaque forming assays and the like). The B cells may then be usedas source of a SOST specific antibody-encoding polynucleotide that canbe cloned and/or modified by recombinant molecular biology techniquesknown in the art and described herein.

A “biological sample” as used herein refers in certain embodiments to asample containing at least one antibody specific for a SOST polypeptide,and a biological sample may be provided by obtaining a blood sample(from which serum or plasma may be prepared), biopsy specimen, tissueexplant, organ culture, or any other tissue or cell preparation from asubject or a biological source. A sample may further refer to a tissueor cell preparation in which the morphological integrity or physicalstate has been disrupted, for example, by dissection, dissociation,solubilization, fractionation, homogenization, biochemical or chemicalextraction, pulverization, lyophilization, sonication, or any othermeans for processing a sample derived from a subject or biologicalsource. The subject or biological source may be a human or non-humananimal, a primary cell culture (e.g., B cells immunized in vitro), orculture adapted cell line including but not limited to geneticallyengineered cell lines that may contain chromosomally integrated orepisomal recombinant nucleic acid sequences, immortalized orimmortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell lines,and the like.

SOST peptide immunogens may also be prepared by synthesizing a series ofpeptides that, in total, represent the entire polypeptide sequence of aSOST polypeptide and that each have a portion of the SOST amino acidsequence in common with another peptide in the series. This overlappingportion would preferably be at least four amino acids, and morepreferably 5, 6, 7, 8, 9, or 10 amino acids. Each peptide may be used toimmunize an animal, the sera collected from the animal, and tested in anassay to identify which animal is producing antibodies that impair orblock binding of SOST to a TGF-beta protein. Antibodies are thenprepared from such identified immunized animals according to methodsknown in the art and described herein.

Peptides, polypeptides, and other non-peptide molecules thatspecifically bind to a TGF-beta binding protein such as SOST arecontemplated by this invention. As used herein, a molecule is said to“specifically bind” to a TGF-beta binding protein if it reacts at adetectable level with the TGF-beta binding protein, but does not reactdetectably with peptides and polypeptides containing an unrelatedsequence, or a sequence of a different TGF-beta binding protein.Preferred binding molecules include antibodies, which may be, forexample, polyclonal, monoclonal, single chain, chimeric, anti-idiotypic,or CDR-grafted immunoglobulins, or fragments thereof, such asproteolytically generated or recombinantly produced immunoglobulinF(ab′)₂, Fab, Fab′ Fv, and Fd fragments.

Particularly useful are anti-TGF-beta binding-protein antibodies that“specifically bind” TGF-beta binding-protein of SEQ ID NOs: 1, 20, 58,60, 62, 64, 66, or 68, but not to other TGF-beta binding-proteins suchas Dan, Cerberus, SCGF, or Gremlin. Antibodies are understood tospecifically bind a TGF-beta binding-protein, or a specific TGF-betafamily member, if they bind with a K_(a) of greater than or equal to 10⁴M⁻¹, more preferably greater than or equal to about 10⁵ M⁻¹, morepreferably greater than or equal to about 10⁶ M⁻¹, still more preferablygreater than or equal to 10⁷ M⁻¹, and still more preferably greater thanor equal to 10⁸ M⁻¹, and do not bind to other TGF-beta binding-proteins.Affinity of an antibody for its cognate antigen is also commonlyexpressed as a dissociation constant K_(D), and an anti-SOST antibodyspecifically binds to a TGF-beta family member if it binds with a K_(D)of less than or equal to 10⁻⁴ M, more preferably less than or equal toabout 10⁻⁵ M, more preferably less than or equal to about 10⁻⁶ M, stillmore preferably less than or equal to 10⁻⁷ M, and still more preferablyless than or equal to 10⁻⁸ M. Furthermore, antibodies of the presentinvention preferably block, impair, or inhibit (e.g., decrease withstatistical significance) binding of a TGF-beta binding-protein to aTGF-beta family member.

The affinity of an antibody or binding partner, as well as the extent towhich an antibody inhibits binding can be readily determined by one ofordinary skill in the art using conventional techniques, for examplethose described by Scatchard et al. (Ann. N.Y. Acad. Sci. 51:660-672,(1949)) or by surface plasmon resonance (SPR; BIAcore, Biosensor,Piscataway, N.J.). For surface plasmon resonance, target molecules areimmobilized on a solid phase and exposed to ligands in a mobile phaserunning along a flow cell. If ligand binding to the immobilized targetoccurs, the local refractive index changes, leading to a change in SPRangle, which can be monitored in real time by detecting changes in theintensity of the reflected light. The rates of change of the SPR signalcan be analyzed to yield apparent rate constants for the association anddissociation phases of the binding reaction. The ratio of these valuesgives the apparent equilibrium constant (affinity) (see, e.g., Wolff etal., Cancer Res. 53:2560-65 (1993)).

An antibody according to the present invention may belong to anyimmunoglobulin class, for example IgG, IgE, IgM, IgD, or IgA. It may beobtained from or derived from an animal, for example, fowl (e.g.,chicken) and mammals, which includes but is not limited to a mouse, rat,hamster, rabbit, or other rodent, a cow, horse, sheep, goat, camel,human, or other primate. The antibody may be an internalising antibody.

Methods well known in the art may be used to generate antibodies,polyclonal antisera, or monoclonal antibodies that are specific for aTGF-beta binding protein such as SOST. Antibodies also may be producedas genetically engineered immunoglobulins (Ig) or Ig fragments designedto have desirable properties. For example, by way of illustration andnot limitation, antibodies may include a recombinant IgG that is achimeric fusion protein having at least one variable (V) region domainfrom a first mammalian species and at least one constant region domainfrom a second, distinct mammalian species. Most commonly, a chimericantibody has murine variable region sequences and human constant regionsequences. Such a murine/human chimeric immunoglobulin may be“humanized” by grafting the complementarity determining regions (CDRs)derived from a murine antibody, which confer binding specificity for anantigen, into human-derived V region framework regions and human-derivedconstant regions. Fragments of these molecules may be generated byproteolytic digestion, or optionally, by proteolytic digestion followedby mild reduction of disulfide bonds and alkylation. Alternatively, suchfragments may also be generated by recombinant genetic engineeringtechniques.

Certain preferred antibodies are those antibodies that inhibit or blocka TGF-beta binding protein activity within an in vitro assay, asdescribed herein. Binding properties of an antibody to a TGF-betabinding protein may generally be assessed using immunodetection methodsincluding, for example, an enzyme-linked immunosorbent assay (ELISA),immunoprecipitation, immunoblotting, countercurrentimmunoelectrophoresis, radioimmunoassays, dot blot assays, inhibition orcompetition assays, and the like, which may be readily performed bythose having ordinary skill in the art (see, e.g., U.S. Pat. Nos.4,376,110 and 4,486,530; Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988)).

An immunogen may be comprised of cells expressing a TGF-beta bindingprotein such as a SOST polypeptide, purified or partially purified SOSTpolypeptide, or variants or fragments (i.e., peptides) thereof, orpeptides derived from a SOST polypeptide. Such peptides may be generatedby proteolytic cleavage of a larger polypeptide, by recombinantmolecular methodologies, or may be chemically synthesized. For instance,nucleic acid sequences encoding SOST polypeptide are provided herein,such that those skilled in the art may routinely prepare SOSTpolypeptide for use as immunogens. Peptides may be chemicallysynthesized by methods as described herein and known in the art.Alternatively, peptides may be generated by proteolytic cleavage of aSOST polypeptide, and individual peptides isolated by methods known inthe art such as polyacrylamide gel electrophoresis or any number ofliquid chromatography or other separation methods. Peptides useful asimmunogens typically may have an amino acid sequence of at least 4 or 5consecutive amino acids from a SOST polypeptide amino acid sequence suchas those described herein, and preferably have at least 6, 7, 8, 9, 10,11, 12, 14, 15, 16, 18, 19 or 20 consecutive amino acids of a SOSTpolypeptide. Certain other preferred peptide immunogens comprise atleast 6 but no more than 12 or more consecutive amino acids of a SOSTpolypeptide sequence, and other preferred peptide immunogens comprise atleast 20 but no more than 75 consecutive amino acids, and otherpreferred peptide immunogens comprise at least 21 but no more than 50consecutive amino acids of a SOST polypeptide. Other preferred peptideimmunogens comprise 21-25, 26-30, 31-35, 36-40, 41-50, or any wholeinteger number of amino acids between and including 21 and 100consecutive amino acids, and between 100 and 190 consecutive amino acidsof a SOST polypeptide sequence.

Polyclonal antibodies that bind specifically to SOST can be preparedusing methods described herein and well-known to persons skilled in theart (see, for example, Green et al., “Production of PolyclonalAntisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (HumanaPress 1992); Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory (1988); Williams et al., “Expression of foreignproteins in E. coli using plasmid vectors and purification of specificpolyclonal antibodies,” in DNA Cloning 2: Expression Systems, 2ndEdition, Glover et al. (eds.), page 15 (Oxford University Press 1995)).Antibodies to a TGF-beta binding-protein can be obtained, for example,by immunizing an animal with the TGF-beta binding-protein product of anexpression vector, or by immunizing with an isolated TGF-beta bindingprotein or a peptide fragment thereof, as described herein. Althoughpolyclonal antibodies are typically raised in animals such as rats,mice, rabbits, goats, cattle, or sheep, an anti-TGF-beta binding-proteinantibody of the present invention may also be obtained from a subhumanprimate. General techniques for raising diagnostically andtherapeutically useful antibodies in baboons may be found, for example,in WO 91/11465 (1991) and in Losman et al., Int. J. Cancer 46:310, 1990.

Preparation of an Immunogen for Injection into Animals May Includecovalent coupling of the TGF-beta binding protein (or variant orfragment thereof), to another immunogenic protein, for example, acarrier protein such as keyhole limpet hemocyanin (KLH) or bovine serumalbumin (BSA) or the like. A polypeptide or peptide immunogen mayinclude one or more additional amino acids at either the N-terminal orC-terminal end that facilitate the conjugation procedure (e.g., theaddition of a cysteine to facilitate conjugation of a peptide to KLH).Other amino acid residues within a polypeptide or peptide may besubstituted to prevent conjugation at that particular amino acidposition to a carrier polypeptide (e.g., substituting a serine residuefor cysteine at internal positions of a polypeptide/peptide) or may besubstituted to facilitate solubility or to increase immunogenicity.

TGF-beta binding protein peptide, polypeptide, or TGF-beta bindingprotein-expressing cells to be used as an immunogen may be emulsified inan adjuvant, for example, Freund's complete or incomplete adjuvant orthe Ribi Adjuvant System (Corixa Corporation, Seattle, Wash.). See also,e.g., Harlow et al., supra. The immunogen may be injected into theanimal via any number of different routes, including intraperitoneally,intramuscularly, intraocularly, intradermally, or subcutaneously. Ingeneral, after the first injection, animals receive one or more boosterimmunizations according to a preferred schedule that may vary accordingto, inter alia, the antigen, the adjuvant (if any), and/or theparticular animal species. The immune response may be monitored byperiodically bleeding the animal and preparing and analyzing sera in animmunoassay, such as an ELISA or Ouchterlony diffusion assay, or thelike, to determine the specific antibody titer. Once an adequateantibody titer is established, the animals may be bled periodically toaccumulate the polyclonal antisera or may be exsanguinated. Polyclonalantibodies that bind specifically to the TGF-beta binding protein orpeptide may then be purified from such antisera, for example, byaffinity chromatography using protein A. Alternatively, affinitychromatography may be performed wherein the TGF-beta binding protein orpeptide or an antibody specific for an Ig constant region of theparticular immunized animal species is immobilized on a suitable solidsupport.

Antibodies for use in the invention include monoclonal antibodies thatare prepared by conventional immunization and cell fusion procedures asdescribed herein an known in the art. Monoclonal anti-TGF-betabinding-protein antibodies may be generated using a variety oftechniques. Monoclonal antibodies that bind to specific antigens may beobtained by methods known to those skilled in the art (see, for example,Kohler et al., Nature 256:495, 1975; Coligan et al. (eds.), CurrentProtocols in Immunology, 1:2.5.1-2.6.7 (John Wiley & Sons 1991); U.S.Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; MonoclonalAntibodies, Hybridomas: A New Dimension in Biological Analyses, PlenumPress, Kennett, McKeam, and Bechtol (eds.) (1980); and Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress (1988); Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)). Antibody fragments may be derived therefrom using anysuitable standard technique such as proteolytic digestion, oroptionally, by proteolytic digestion (for example, using papain orpepsin) followed by mild reduction of disulfide bonds and alkylation.Alternatively, such fragments may also be generated by recombinantgenetic engineering techniques.

Briefly, monoclonal antibodies can be obtained by injecting an animal,for example, a rat, hamster, or preferably a mouse, with an immunogencomprising a TGF-beta binding-protein gene product, or peptide fragmentthereof, according to methods known in the art and described herein. Thepresence of specific antibody production may be monitored after theinitial injection (injections may be administered by any one of severalroutes as described herein for generation of polyclonal antibodies)and/or after a booster injection by obtaining a serum sample anddetecting the presence of an antibody that binds to a TGF-betabinding-protein or peptide using any one of several immunodetectionmethods known in the art and described herein. From animals producingantibodies that bind to the TGF-beta binding protein, lymphoid cells,most commonly cells from the spleen or lymph node, are removed to obtainB-lymphocytes. The B-lymphocytes are then fused with a drug-sensitizedmyeloma cell fusion partner, preferably one that is syngeneic with theimmunized animal and that optionally has other desirable properties(e.g., inability to express endogenous Ig gene products, e.g., P3×63-Ag8.653 (ATCC No. CRL 1580); NS0, SP20) to produce hybridomas, which areimmortal eukaryotic cell lines. The lymphoid (e.g., spleen) cells andthe myeloma cells may be combined for a few minutes with a membranefusion-promoting agent, such as polyethylene glycol or a nonionicdetergent, and then plated at low density on a selective medium thatsupports the growth of hybridoma cells but not unfused myeloma cells. Apreferred selection media is HAT (hypoxanthine, aminopterin, thymidine).After a sufficient time, usually about one to two weeks, colonies ofcells are observed. Single colonies are isolated, and antibodiesproduced by the cells may be tested for binding activity to the TGF-betabinding protein, or variant or fragment thereof, using any one of avariety of immunoassays known in the art and described herein.Hybridomas producing monoclonal antibodies with high affinity andspecificity for SOST are preferred. Hybridomas that produce monoclonalantibodies that specifically bind to a TGF-beta binding protein orvariant or fragment thereof are therefore contemplated by the presentinvention. The hybridomas are cloned (e.g., by limited dilution cloningor by soft agar plaque isolation) and positive clones that produce anantibody specific to the antigen are selected and cultured. Antibodiesthat block, inhibit, or impair binding of the TGF-beta binding proteinto a TGF-beta family member are preferred.

The monoclonal antibodies from the hybridoma cultures may be isolatedfrom the supernatants of hybridoma cultures. An alternative method forproduction of a murine monoclonal antibody is to inject the hybridomacells into the peritoneal cavity of a syngeneic mouse, for example, amouse that has been treated (e.g., pristane-primed) to promote formationof ascites fluid containing the monoclonal antibody. Monoclonalantibodies can be isolated and purified by a variety of well-establishedtechniques. Such isolation techniques include affinity chromatographywith Protein-A Sepharose, size-exclusion chromatography, andion-exchange chromatography (see, for example, Coligan at pages2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., “Purification ofImmunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages79-104 (The Humana Press, Inc. 1992)). Monoclonal antibodies may bepurified by affinity chromatography using an appropriate ligand selectedbased on particular properties of the antibody (e.g., heavy or lightchain isotype, binding specificity, etc.). Examples of a suitableligand, immobilized on a solid support, include Protein A, Protein G, ananti-constant region (light chain or heavy chain) antibody, ananti-idiotype antibody, and a TGF-beta binding protein, or fragment orvariant thereof.

In addition, an anti-TGF-beta binding-protein antibody of the presentinvention may be a human monoclonal antibody. Human monoclonalantibodies may be generated by any number of techniques with which thosehaving ordinary skill in the art will be familiar. Such methods include,but are not limited to, Epstein Barr Virus (EBV) transformation of humanperipheral blood cells (e.g., containing B lymphocytes), in vitroimmunization of human B cells, fusion of spleen cells from immunizedtransgenic mice carrying inserted human immunoglobulin genes, isolationfrom human immunoglobulin V region phage libraries, or other proceduresas known in the art and based on the disclosure herein. For example,human monoclonal antibodies may be obtained from transgenic mice thathave been engineered to produce specific human antibodies in response toantigenic challenge. Methods for obtaining human antibodies fromtransgenic mice are described, for example, by Green et al., NatureGenet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al.,Int. Immun. 6:579, 1994; U.S. Pat. No. 5,877,397; Bruggemann et al.,1997 Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al., 1995 Ann. N.Y.Acad. Sci. 764:525-35. In this technique, elements of the human heavyand light chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. (See also Bruggemann etal., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, humanimmunoglobulin transgenes may be mini-gene constructs, or transloci onyeast artificial chromosomes, which undergo B cell-specific DNArearrangement and hypermutation in the mouse lymphoid tissue. Humanmonoclonal antibodies may be obtained by immunizing the transgenic mice,which may then produce human antibodies specific for the antigen.Lymphoid cells of the immunized transgenic mice can be used to producehuman antibody-secreting hybridomas according to the methods describedherein. Polyclonal sera containing human antibodies may also be obtainedfrom the blood of the immunized animals.

Another method for generating human TGF-beta binding protein specificmonoclonal antibodies includes immortalizing human peripheral bloodcells by EBV transformation. See, e.g., U.S. Pat. No. 4,464,456. Such animmortalized B cell line (or lymphoblastoid cell line) producing amonoclonal antibody that specifically binds to a TGF-beta bindingprotein (or a variant or fragment thereof) can be identified byimmunodetection methods as provided herein, for example, an ELISA, andthen isolated by standard cloning techniques. The stability of thelymphoblastoid cell line producing an anti-TGF-beta binding proteinantibody may be improved by fusing the transformed cell line with amurine myeloma to produce a mouse-human hybrid cell line according tomethods known in the art (see, e.g., Glasky et al., Hybridoma 8:377-89(1989)). Still another method to generate human monoclonal antibodies isin vitro immunization, which includes priming human splenic B cells withantigen, followed by fusion of primed B cells with a heterohybrid fusionpartner. See, e.g., Boerner et al., 1991 J. Immunol. 147:86-95.

In certain embodiments, a B cell that is producing an anti-SOST antibodyis selected and the light chain and heavy chain variable regions arecloned from the B cell according to molecular biology techniques knownin the art (WO 92/02551; U.S. Pat. No. 5,627,052; Babcook et al., Proc.Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. PreferablyB cells from an immunized animal are isolated from the spleen, lymphnode, or peripheral blood sample by selecting a cell that is producingan antibody that specifically binds to SOST. B cells may also beisolated from humans, for example, from a peripheral blood sample.Methods for detecting single B cells that are producing an antibody withthe desired specificity are well known in the art, for example, byplaque formation, fluorescence-activated cell sorting, in vitrostimulation followed by detection of specific antibody, and the like.Methods for selection of specific antibody producing B cells include,for example, preparing a single cell suspension of B cells in soft agarthat contains SOST or a peptide fragment thereof. Binding of thespecific antibody produced by the B cell to the antigen results in theformation of a complex, which may be visible as an immunoprecipitate.After the B cells producing the specific antibody are selected, thespecific antibody genes may be cloned by isolating and amplifying DNA ormRNA according to methods known in the art and described herein.

For particular uses, fragments of anti-TGF-beta binding proteinantibodies may be desired. Antibody fragments, F(ab′)₂, Fab, Fab′, Fv,Fc, Fd, retain the antigen binding site of the whole antibody andtherefore bind to the same epitope. These antigen-binding fragmentsderived from an antibody can be obtained, for example, by proteolytichydrolysis of the antibody, for example, pepsin or papain digestion ofwhole antibodies according to conventional methods. As an illustration,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent to produce 3.5S Fab′monovalent fragments. Optionally, the cleavage reaction can be performedusing a blocking group for the sulfhydryl groups that result fromcleavage of disulfide linkages. As an alternative, an enzymatic cleavageusing papain produces two monovalent Fab fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,331,647, Nisonoff et al., Arch. Biochem. Biophys. 89:230,1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., in Methods inEnzymology 1:422 (Academic Press 1967); and by Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4. Other methods for cleaving antibodies,such as separating heavy chains to form monovalent light-heavy chainfragments (Fd), further cleaving of fragments, or other enzymatic,chemical, or genetic techniques may also be used, so long as thefragments bind to the antigen that is recognized by the intact antibody.

An antibody fragment may also be any synthetic or genetically engineeredprotein that acts like an antibody in that it binds to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (scFvproteins), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region. The antibody of thepresent invention preferably comprises at least one variable regiondomain. The variable region domain may be of any size or amino acidcomposition and will generally comprise at least one hypervariable aminoacid sequence responsible for antigen binding and which is adjacent toor in frame with one or more framework sequences. In general terms, thevariable (V) region domain may be any suitable arrangement ofimmunoglobulin heavy (V_(H)) and/or light (V_(L)) chain variabledomains. Thus, for example, the V region domain may be monomeric and bea V_(H) or V_(L) domain, which is capable of independently bindingantigen with acceptable affinity. Alternatively, the V region domain maybe dimeric and contain V_(H)-V_(H), V_(H)-V_(L), or V_(L)-V_(L), dimers.Preferably, the V region dimer comprises at least one V_(H) and at leastone V_(L) chain that are non-covalently associated (hereinafter referredto as F_(V)). If desired, the chains may be covalently coupled eitherdirectly, for example via a disulfide bond between the two variabledomains, or through a linker, for example a peptide linker, to form asingle chain Fv (scF_(V)).

The variable region domain may be any naturally occurring variabledomain or an engineered version thereof. By engineered version is meanta variable region domain that has been created using recombinant DNAengineering techniques. Such engineered versions include those created,for example, from a specific antibody variable region by insertions,deletions, or changes in or to the amino acid sequences of the specificantibody. Particular examples include engineered variable region domainscontaining at least one CDR and optionally one or more framework aminoacids from a first antibody and the remainder of the variable regiondomain from a second antibody.

The variable region domain may be covalently attached at a C-terminalamino acid to at least one other antibody domain or a fragment thereof.Thus, for example, a V_(H) domain that is present in the variable regiondomain may be linked to an immunoglobulin C_(H)1 domain, or a fragmentthereof. Similarly a V_(L) domain may be linked to a C_(K) domain or afragment thereof. In this way, for example, the antibody may be a Fabfragment wherein the antigen binding domain contains associated V_(H)and V_(L) domains covalently linked at their C-termini to a CH1 andC_(K) domain, respectively. The CH1 domain may be extended with furtheramino acids, for example to provide a hinge region or a portion of ahinge region domain as found in a Fab′ fragment, or to provide furtherdomains, such as antibody CH2 and CH3 domains.

Another form of an antibody fragment is a peptide comprising for asingle complementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing polynucleotides thatencode the CDR of an antibody of interest. Such polynucleotides areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region using mRNA of antibody-producing cells asa template (see, for example, Larrick et al., Methods: A Companion toMethods in Enzymology 2:106, 1991; Courtenay-Luck, “Genetic Manipulationof Monoclonal Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), page 166(Cambridge University Press 1995); and Ward et al., “GeneticManipulation and Expression of Antibodies,” in Monoclonal Antibodies:Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss,Inc. 1995)).

Alternatively, the antibody may be a recombinant or engineered antibodyobtained by the use of recombinant DNA techniques involving themanipulation and re-expression of DNA encoding antibody variable and/orconstant regions. Such DNA is known and/or is readily available from DNAlibraries including for example phage-antibody libraries (see Chiswelland McCafferty, Tibtech. 10:80-84 (1992)) or if desired can besynthesized. Standard molecular biology and/or chemistry procedures maybe used to sequence and manipulate the DNA, for example, to introducecodons to create cysteine residues, or to modify, add or delete otheramino acids or domains as desired.

Chimeric antibodies, specific for a TGF-beta binding protein, and whichinclude humanized antibodies, may also be generated according to thepresent invention. A chimeric antibody has at least one constant regiondomain derived from a first mammalian species and at least one variableregion domain derived from a second, distinct mammalian species (see,e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-55 (1984)).In preferred embodiments, a chimeric antibody may be constructed bycloning the polynucleotide sequence that encodes at least one variableregion domain derived from a non-human monoclonal antibody, such as thevariable region derived from a murine, rat, or hamster monoclonalantibody, into a vector containing a nucleotide sequence that encodes atleast one human constant region (see, e.g., Shin et al., MethodsEnzymol. 178:459-76 (1989); Walls et al., Nucleic Acids Res. 21:2921-29(1993)). By way of example, the polynucleotide sequence encoding thelight chain variable region of a murine monoclonal antibody may beinserted into a vector containing a nucleotide sequence encoding thehuman kappa light chain constant region sequence. In a separate vector,the polynucleotide sequence encoding the heavy chain variable region ofthe monoclonal antibody may be cloned in frame with sequences encoding ahuman IgG constant region, for example, the human IgG1 constant region.The particular human constant region selected may depend upon theeffector functions desired for the particular antibody (e.g., complementfixing, binding to a particular Fc receptor, etc.). Preferably, theconstructed vectors will be transfected into eukaryotic cells for stableexpression of the chimeric antibody. Another method known in the art forgenerating chimeric antibodies is homologous recombination (e.g., U.S.Pat. No. 5,482,856).

A non-human/human chimeric antibody may be further geneticallyengineered to create a “humanized” antibody. Such a humanized antibodymay comprise a plurality of CDRs derived from an immunoglobulin of anon-human mammalian species, at least one human variable frameworkregion, and at least one human immunoglobulin constant region. Usefulstrategies for designing humanized antibodies may include, for exampleby way of illustration and not limitation, identification of humanvariable framework regions that are most homologous to the non-humanframework regions of the chimeric antibody. Without wishing to be boundby theory, such a strategy may increase the likelihood that thehumanized antibody will retain specific binding affinity for a TGF-betabinding protein, which in some preferred embodiments may besubstantially the same affinity for a TGF-beta binding protein orvariant or fragment thereof, and in certain other preferred embodimentsmay be a greater affinity for TGF-beta binding protein. See, e.g., Joneset al., 1986 Nature 321:522-25; Riechmann et al., 1988 Nature332:323-27. Designing such a humanized antibody may therefore includedetermining CDR loop conformations and structural determinants of thenon-human variable regions, for example, by computer modeling, and thencomparing the CDR loops and determinants to known human CDR loopstructures and determinants. See, e.g., Padlan et al., 1995 FASEB9:133-39; Chothia et al., 1989 Nature, 342:377-383. Computer modelingmay also be used to compare human structural templates selected bysequence homology with the non-human variable regions. See, e.g.,Bajorath et al., 1995 Ther. Immunol. 2:95-103; EP-0578515-A3. Ifhumanization of the non-human CDRs results in a decrease in bindingaffinity, computer modeling may aid in identifying specific amino acidresidues that could be changed by site-directed or other mutagenesistechniques to partially, completely or supra-optimally (i.e., increaseto a level greater than that of the non-humanized antibody) restoreaffinity. Those having ordinary skill in the art are familiar with thesetechniques, and will readily appreciate numerous variations andmodifications to such design strategies.

One such method for preparing a humanized antibody is called veneering.As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site thatretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.,Ann. Rev. Biochem. 59:439-73, 1990. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residuesthat are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein). Solvent accessibilities of V regionamino acids can be deduced from the known three-dimensional structurefor human and murine antibody fragments. Initially, the FRs of thevariable domains of an antibody molecule of interest are compared withcorresponding FR sequences of human variable domains obtained from theabove-identified sources. The most homologous human V regions are thencompared residue by residue to corresponding murine amino acids. Theresidues in the murine FR that differ from the human counterpart arereplaced by the residues present in the human moiety using recombinanttechniques well known in the art. Residue switching is only carried outwith moieties which are at least partially exposed (solvent accessible),and care is exercised in the replacement of amino acid residues that mayhave a significant effect on the tertiary structure of V region domains,such as proline, glycine, and charged amino acids.

In this manner, the resultant “veneered” antigen-binding sites are thusdesigned to retain the rodent CDR residues, the residues substantiallyadjacent to the CDRs, the residues identified as buried or mostly buried(solvent inaccessible), the residues believed to participate innon-covalent (e.g., electrostatic and hydrophobic) contacts betweenheavy and light chain domains, and the residues from conservedstructural regions of the FRs which are believed to influence the“canonical” tertiary structures of the CDR loops. These design criteriaare then used to prepare recombinant nucleotide sequences that combinethe CDRs of both the heavy and light chain of a antigen-binding siteinto human-appearing FRs that can be used to transfect mammalian cellsfor the expression of recombinant human antibodies that exhibit theantigen specificity of the rodent antibody molecule.

An additional method for selecting antibodies that specifically bind toa TGF-beta binding protein or variant or fragment thereof is by phagedisplay. See, e.g., Winter et al., 1994 Annu. Rev. Immunol. 12:433-55;Burton et al., 1994 Adv. Immunol. 57:191-280. Human or murineimmunoglobulin variable region gene combinatorial libraries may becreated in phage vectors that can be screened to select Ig fragments(Fab, Fv, sFv, or multimers thereof) that bind specifically to TGF-betabinding protein or variant or fragment thereof. See, e.g., U.S. Pat. No.5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc.Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategiesin Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad.Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol.227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and referencescited therein. For example, a library containing a plurality ofpolynucleotide sequences encoding Ig variable region fragments may beinserted into the genome of a filamentous bacteriophage, such as M13 ora variant thereof, in frame with the sequence encoding a phage coatprotein. A fusion protein may be a fusion of the coat protein with thelight chain variable region domain and/or with the heavy chain variableregion domain. According to certain embodiments, immunoglobulin Fabfragments may also be displayed on a phage particle (see, e.g., U.S.Pat. No. 5,698,426).

Heavy and light chain immunoglobulin cDNA expression libraries may alsobe prepared in lambda phage, for example, using λImmunoZap™(H) and λImmunoZap™(L) vectors (Stratagene, La Jolla, Calif.). Briefly, mRNA isisolated from a B cell population, and used to create heavy and lightchain immunoglobulin cDNA expression libraries in the λImmunoZap(H) andλImmunoZap(L) vectors. These vectors may be screened individually orco-expressed to form Fab fragments or antibodies (see Huse et al.,supra; see also Sastry et al., supra). Positive plaques may subsequentlybe converted to a non-lytic plasmid that allows high level expression ofmonoclonal antibody fragments from E. coli.

Similarly, portions or fragments, such as Fab and Fv fragments, ofantibodies may also be constructed using conventional enzymaticdigestion or recombinant DNA techniques to incorporate the variableregions of a gene that encodes a antibody specific for a TGF-betabinding protein. Within one embodiment, in a hybridoma the variableregions of a gene expressing a monoclonal antibody of interest areamplified using nucleotide primers. These primers may be synthesized byone of ordinary skill in the art, or may be purchased from commerciallyavailable sources. (See, e.g., Stratagene (La Jolla, Calif.), whichsells primers for mouse and human variable regions including, amongothers, primers for V_(Ha), V_(Hb), V_(Hc), V_(Hd), C_(H1), V_(L) andC_(L) regions.) These primers may be used to amplify heavy or lightchain variable regions, which may then be inserted into vectors such asImmunoZAP™ H or ImmunoZAP™ L (Stratagene), respectively. These vectorsmay then be introduced into E. coli, yeast, or mammalian-based systemsfor expression. Large amounts of a single-chain protein containing afusion of the V_(H) and V_(L) domains may be produced using thesemethods (see Bird et al., Science 242:423-426, 1988). In addition, suchtechniques may be used to humanize a non-human antibody V region withoutaltering the binding specificity of the antibody.

In certain particular embodiments of the invention, combinatorial phagelibraries may also be used for humanization of non-human variableregions. See, e.g., Rosok et al., 1996 J. Biol. Chem. 271:22611-18;Rader et al., 1998 Proc. Natl. Acad. Sci. USA 95:8910-15. A phagelibrary may be screened to select an Ig variable region fragment ofinterest by immunodetection methods known in the art and describedherein, and the DNA sequence of the inserted immunoglobulin gene in thephage so selected may be determined by standard techniques. See,Sambrook et al., 2001 Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press. The selected Ig-encoding sequence may then becloned into another suitable vector for expression of the Ig fragmentor, optionally, may be cloned into a vector containing Ig constantregions, for expression of whole immunoglobulin chains.

In certain other embodiments, the invention contemplates SOST-specificantibodies that are multimeric antibody fragments. Useful methodologiesare described generally, for example in Hayden et al. 1997, Curr Opin.Immunol. 9:201-12; Coloma et al., 1997 Nat. Biotechnol. 15:159-63. Forexample, multimeric antibody fragments may be created by phagetechniques to form miniantibodies (U.S. Pat. No. 5,910,573) or diabodies(Holliger et al., 1997, Cancer Immunol. Immunother. 45:128-130).

In certain embodiments of the invention, an antibody specific for SOSTmay be an antibody that is expressed as an intracellular protein. Suchintracellular antibodies are also referred to as intrabodies and maycomprise an Fab fragment, or preferably comprise a scFv fragment (see,e.g., Lecerf et al., Proc. Natl. Acad. Sci. USA 98:4764-49 (2001). Theframework regions flanking the CDR regions can be modified to improveexpression levels and solubility of an intrabody in an intracellularreducing environment (see, e.g., Worn et al., J. Biol. Chem.275:2795-803 (2000). An intrabody may be directed to a particularcellular location or organelle, for example by constructing a vectorthat comprises a polynucleotide sequence encoding the variable regionsof an intrabody that may be operatively fused to a polynucleotidesequence that encodes a particular target antigen within the cell (see,e.g., Graus-Porta et al., Mol. Cell. Biol. 15:1182-91 (1995); Lener etal., Eur. J. Biochem. 267:1196-205 (2000)). An intrabody may beintroduced into a cell by a variety of techniques available to theskilled artisan including via a gene therapy vector, or a lipid mixture(e.g., Provectin™ manufactured by Imgenex Corporation, San Diego,Calif.), or according to photochemical internalization methods.

Introducing amino acid mutations into an immunoglobulin moleculespecific for a TGF-beta binding protein may be useful to increase thespecificity or affinity for TGF-beta binding protein or to alter aneffector function. Immunoglobulins with higher affinity for TGF-betabinding protein may be generated by site-directed mutagenesis ofparticular residues. Computer assisted three-dimensional molecularmodeling may be employed to identify the amino acid residues to bechanged, in order to improve affinity for the TGF-beta binding protein.See, e.g., Mountain et al., 1992, Biotechnol. Genet. Eng. Rev. 10:1-142.Alternatively, combinatorial libraries of CDRs may be generated in M13phage and screened for immunoglobulin fragments with improved affinity.See, e.g., Glaser et al., 1992, J. Immunol. 149:3903-3913; Barbas etal., 1994 Proc. Natl. Acad. Sci. USA 91:3809-13; U.S. Pat. No.5,792,456.

Effector functions may also be altered by site-directed mutagenesis.See, e.g., Duncan et al., 1988 Nature 332:563-64; Morgan et al., 1995Immunology 86:319-24; Eghtedarzedeh-Kondri et al., 1997 Biotechniques23:830-34. For example, mutation of the glycosylation site on the Fcportion of the immunoglobulin may alter the ability of theimmunoglobulin to fix complement. See, e.g., Wright et al., 1997 TrendsBiotechnol. 15:26-32. Other mutations in the constant region domains mayalter the ability of the immunoglobulin to fix complement, or to effectantibody-dependent cellular cytotoxicity. See, e.g., Duncan et al., 1988Nature 332:563-64; Morgan et al., 1995 Immunology 86:319-24; Sensel etal., 1997 Mol. Immunol. 34:1019-29.

According to certain embodiments, non-human, human, or humanized heavychain and light chain variable regions of any of the Ig moleculesdescribed herein may be constructed as single chain Fv (scFv)polypeptide fragments (single chain antibodies). See, e.g., Bird et al.,1988 Science 242:423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. USA85:5879-5883. Multi-functional scFv fusion proteins may be generated bylinking a polynucleotide sequence encoding an scFv polypeptide in-framewith at least one polynucleotide sequence encoding any of a variety ofknown effector proteins. These methods are known in the art, and aredisclosed, for example, in EP-BI-0318554, U.S. Pat. No. 5,132,405, U.S.Pat. No. 5,091,513, and U.S. Pat. No. 5,476,786. By way of example,effector proteins may include immunoglobulin constant region sequences.See, e.g., Hollenbaugh et al., 1995 J. Immunol. Methods 188:1-7. Otherexamples of effector proteins are enzymes. As a non-limiting example,such an enzyme may provide a biological activity for therapeuticpurposes (see, e.g., Siemers et al., 1997 Bioconjug. Chem. 8:510-19), ormay provide a detectable activity, such as horseradishperoxidase-catalyzed conversion of any of a number of well-knownsubstrates into a detectable product, for diagnostic uses. Still otherexamples of scFv fusion proteins include Ig-toxin fusions, orimmunotoxins, wherein the scFv polypeptide is linked to a toxin.

The scFv or any antibody fragment described herein may, in certainembodiments, be fused to peptide or polypeptide domains that permitsdetection of specific binding between the fusion protein and antigen(e.g., a TGF-beta binding protein). For example, the fusion polypeptidedomain may be an affinity tag polypeptide for detecting binding of thescFv fusion protein to a TGF-beta binding protein by any of a variety oftechniques with which those skilled in the art will be familiar.Examples of a peptide tag, include avidin, streptavidin or His (e.g.,polyhistidine). Detection techniques may also include, for example,binding of an avidin or streptavidin fusion protein to biotin or to abiotin mimetic sequence (see, e.g., Luo et al., 1998 J. Biotechnol.65:225 and references cited therein), direct covalent modification of afusion protein with a detectable moiety (e.g., a labeling moiety),non-covalent binding of the fusion protein to a specific labeledreporter molecule, enzymatic modification of a detectable substrate by afusion protein that includes a portion having enzyme activity, orimmobilization (covalent or non-covalent) of the fusion protein on asolid-phase support. Other useful affinity polypeptides for constructionof scFv fusion proteins may include streptavidin fusion proteins, asdisclosed, for example, in WO 89/03422, U.S. Pat. No. 5,489,528, U.S.Pat. No. 5,672,691, WO 93/24631, U.S. Pat. No. 5,168,049, U.S. Pat. No.5,272,254; avidin fusion proteins (see, e.g., EP 511,747); an enzymesuch as glutathione-S-transferase; and Staphylococcus aureus protein Apolypeptide.

The polynucleotides encoding an antibody or fragment thereof thatspecifically bind a TGF-beta binding protein, as described herein, maybe propagated and expressed according to any of a variety of well-knownprocedures for nucleic acid excision, ligation, transformation, andtransfection using any number of known expression vectors. Thus, incertain embodiments expression of an antibody fragment may be preferredin a prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun etal., 1989 Methods Enzymol. 178:497-515). In certain other embodiments,expression of the antibody or a fragment thereof may be preferred in aeukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Pichia pastoris), animal cells (includingmammalian cells) or plant cells. Examples of suitable animal cellsinclude, but are not limited to, myeloma (such as a mouse NSO line),COS, CHO, or hybridoma cells. Examples of plant cells include tobacco,corn, soybean, and rice cells.

One or more replicable expression vectors containing DNA encoding avariable and/or constant region may be prepared and used to transform anappropriate cell line, for example, a non-producing myeloma cell line,such as a mouse NSO line or a bacteria, such as E. coli, in whichproduction of the antibody will occur. In order to obtain efficienttranscription and translation, the DNA sequence in each vector shouldinclude appropriate regulatory sequences, particularly a promoter andleader sequence operatively linked to the variable domain sequence.Particular methods for producing antibodies in this way are generallywell known and routinely used. For example, basic molecular biologyprocedures are described by Maniatis et al. (Molecular Cloning, ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York,1989; see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory,New York, (2001)). DNA sequencing can be performed as described inSanger et al (PNAS 74:5463, (1977)) and the Amersham International plcsequencing handbook, and site directed mutagenesis can be carried outaccording to methods known in the art (Kramer et al., Nucleic Acids Res.12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92 (1985);Kunkel et al., Methods in Enzymol. 154:367-82 (1987); the AnglianBiotechnology Ltd handbook). Additionally, numerous publicationsdescribe techniques suitable for the preparation of antibodies bymanipulation of DNA, creation of expression vectors, and transformationof appropriate cells (Mountain A and Adair, J R in Biotechnology andGenetic Engineering Reviews (ed. Tombs, M P. 10, Chapter 1, 1992,Intercept, Andover, UK); International Patent Specification No. WO91/09967).

In certain embodiments, the antibody according to the invention may haveone or more effector or reporter molecules attached to it. A reportermolecule may be a detectable moiety or label such as an enzyme,cytotoxic agent or other reporter molecule, including a dye,radionuclide, luminescent group, fluorescent group, or biotin, or thelike. The TGF-beta binding protein-specific immunoglobulin or fragmentthereof may be radiolabeled for diagnostic or therapeutic applications.Techniques for radiolabeling of antibodies are known in the art. See,e.g., Adams 1998 In Vivo 12:11-21; Hiltunen 1993 Acta Oncol. 32:831-9.Therapeutic applications are described in greater detail below and mayinclude use of the TGF-beta binding protein specific antibody (orfragment thereof) in conjunction with other therapeutic agents. Theeffector or reporter molecules may be attached to the antibody throughany available amino acid side-chain, terminal amino acid, orcarbohydrate functional group located in the antibody, provided that theattachment or attachment process does not adversely affect the bindingproperties such that the usefulness of the molecule is abrogated.Particular functional groups include, for example any free amino, imino,thiol, hydroxyl, carboxyl, or aldehyde group. Attachment of the antibodyand the effector and/or reporter molecule(s) may be achieved via suchgroups and an appropriate functional group in the effector or reportermolecule. The linkage may be direct or indirect through spacing orbridging groups.

Effector molecules include, for example, antineoplastic agents, toxins(such as enzymatically active toxins of bacterial (such as Pseudomonasaeruginosa exotoxin A) or plant origin and fragments thereof (e.g.,ricin and fragments thereof, plant gelonin, bryodin from Byonia dioica,or the like. See, e.g., Thrush et al., 1996 Annu. Rev. Immunol.14:49-71; Frankel et al., 1996 Cancer Res. 56:926-32). Additionaleffector molecules include biologically active proteins (for example,enzymes), nucleic acids and fragments thereof, naturally occurring andsynthetic polymers, for example, polysaccharides and polyalkylenepolymers such as poly(ethylene glycol) and derivatives thereof,radionuclides, particularly radioiodide, and chelated metals. Suitablereporter groups include chelated metals, fluorescent compounds orcompounds that may be detected by NMR or ESR spectroscopy. Particularlyuseful effector groups are calichaemicin and derivatives thereof (see,for example, South African Patent Specifications Nos. 85/8794, 88/8127and 90/2839).

Numerous other toxins, including chemotherapeutic agents, anti-mitoticagents, antibiotics, inducers of apoptosis (or “apoptogens”, see, e.g.,Green and Reed, 1998, Science 281:1309-1312), or the like, are known tothose familiar with the art, and the examples provided herein areintended to be illustrative without limiting the scope and spirit of theinvention. Particular antineoplastic agents include cytotoxic andcytostatic agents, for example alkylating agents, such as nitrogenmustards (e.g., chlorambucil, melphalan, mechlorethamine,cyclophosphamide, or uracil mustard) and derivatives thereof,triethylenephosphoramide, triethylenethiophosphor-amide, busulphan, orcisplatin; antimetabolites, such as methotrexate, fluorouracil,floxuridine, cytarabine, mercaptopurine, thioguanine, fluoroacetic acidor fluorocitric acid, antibiotics, such as bleomycins (e.g., bleomycinsulphate), doxorubicin, daunorubicin, mitomycins (e.g., mitomycin C),actinomycins (e.g., dactinomycin) plicamycin, calichaemicin andderivatives thereof, or esperamicin and derivatives thereof; mitoticinhibitors, such as etoposide, vincristine or vinblastine andderivatives thereof; alkaloids, such as ellipticine; polyols such astaxicin-I or taxicin-II; hormones, such as androgens (e.g.,dromostanolone or testolactone), progestins (e.g., megestrol acetate ormedroxyprogesterone acetate), estrogens (e.g., dimethylstilbestroldiphosphate, polyestradiol phosphate or estramustine phosphate) orantiestrogens (e.g., tamoxifen); anthraquinones, such as mitoxantrone,ureas, such as hydroxyurea; hydrazines, such as procarbazine; orimidazoles, such as dacarbazine.

Chelated metals useful as effector molecules include chelates of di- ortripositive metals having a coordination number from 2 to 8 inclusive.Particular examples of such metals include technetium (Tc), rhenium(Re), cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb),bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb),gadolinium (Gd), and scandium (Sc). In general the metal is preferably aradionuclide. Particular radionuclides include ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸R,⁵⁸C, ⁶⁰Co, ⁶⁷Cu, ¹⁹⁵Au, ¹⁹⁹Au, ¹¹⁰Ag, ²⁰³ Pb, ²⁰⁶ Bi, ²⁰⁷Bi, ¹¹¹In,⁶⁷Ga, ⁶⁸Ga, ⁸⁸Y, ⁹⁰Y, ¹⁶⁰Tb, ¹⁵³Gd, and ⁴⁷Sc. The chelated metal may befor example one of the above types of metal chelated with any suitablepolydentate chelating agent, for example acyclic or cyclic polyamines,polyethers, (e.g., crown ethers and derivatives thereof), polyamides;porphyrins; and carbocyclic derivatives. In general, the type ofchelating agent will depend on the metal in use. One particularly usefulgroup of chelating agents in conjugates according to the invention,however, comprises acyclic and cyclic polyamines, especiallypolyaminocarboxylic acids, for example diethylenetriaminepentaaceticacid and derivatives thereof, and macrocyclic amines, e.g. cyclictri-aza and tetra-aza derivatives (for example, as described inInternational Patent Specification No. WO 92/22583); and polyamides,especially desferrioxamine and derivatives thereof.

When a thiol group in the antibody is used as the point of attachment,the effector molecule may be attached according to a reaction thatoccurs with a thiol-reactive group present in the effector or reportermolecule. Examples of such groups include an á-halocarboxylic acid orester, such as iodoacetamide, an imide, such as maleimide, a vinylsulphone, or a disulphide. These and other suitable linking proceduresare generally and more particularly described in International PatentSpecifications Nos. WO 93/06231, WO 92/22583, WO 90/091195, and WO89/01476.

The invention also contemplates the generation of anti-idiotypeantibodies that recognize an antibody (or antigen-binding fragmentthereof) that specifically binds to a SOST polypeptide as providedherein, or a variant or fragment thereof. Anti-idiotype antibodies maybe generated as polyclonal antibodies or as monoclonal antibodies by themethods described herein, using an anti-TGF-beta binding proteinantibody (or antigen-binding fragment thereof) as immunogen.Anti-idiotype antibodies or fragments thereof may also be generated byany of the recombinant genetic engineering methods described above, orby phage display selection. An anti-idiotype antibody may react with theantigen binding site of the anti-SOST antibody such that binding of theantibody to a SOST polypeptide is competitively inhibited.Alternatively, an anti-idiotype antibody as provided herein may notcompetitively inhibit binding of an anti-TGF-beta binding proteinantibody to a TGF-beta binding protein.

In certain embodiments of the invention, an antibody that specificallybinds to SOST may be used to detect expression of the SOST polypeptide.In certain particular embodiments, one antibody or a panel of antibodiesmay be exposed to cells that express a SOST polypeptide and expressionof the SOST polypeptide may be determined by detection using anotherSOST specific antibody that binds to a different epitope than theantibody or antibodies first used in the assay.

Assays for Selecting Agents that Increase Bone Density

As discussed above, the present invention provides methods for selectingand/or isolating agents that are capable of increasing bone density. Incertain preferred embodiments, the agent is an antibody thatspecifically binds to SOST (a TGF-beta binding protein) or a variant ora fragment thereof. For example, within one embodiment of the presentinvention, methods are provided for determining whether an agent iscapable of increasing bone mineral content, comprising the steps of (a)contacting or mixing a candidate agent with a TGF-beta binding proteinand a member of the TGF-beta family of proteins and (b) determiningwhether the candidate agent stimulates signaling by the TGF-beta familyof proteins, or impairs or inhibits the binding of the TGF-beta bindingprotein to one or more members of the TGF-beta family of proteins.Within certain embodiments, the agent enhances the ability of TGF-betato function as a positive regulator of mesenchymal cell differentiation.

In a preferred embodiment of the invention, a method is provided foridentifying an antibody that modulates a TGF-beta signaling pathwaycomprising contacting an antibody that specifically binds to a SOSTpolypeptide with a SOST peptide, including but not limited to thepeptides disclosed herein, under conditions and for a time sufficient topermit formation of an antibody plus (+) SOST (antibody/SOST) complexand then detecting the level (e.g., quantifying the amount) of theSOST/antibody complex to determine the presence of an antibody thatmodulates a TGF-beta signaling pathway. The method may be performedusing SPR or any number of different immunoassays known in the art anddisclosed herein, including an ELISA, immunoblot, or the like. ATGF-beta signaling pathway includes a signaling pathway by which a BMPbinds to a type I and a type II receptor on a cell to stimulate orinduce the pathway that modulates bone mineral content. In certainpreferred embodiments of the invention, an antibody that specificallybinds to SOST stimulates or enhances the pathway for increasing bonemineral content. Such an antibody may be identified using the methodsdisclosed herein to detect binding of an antibody to SOST specificpeptides.

The subject invention methods may also be used for identifyingantibodies that impair, inhibit (including competitively inhibit), orprevent binding of a BMP to a SOST polypeptide by detecting whether anantibody binds to SOST peptides that are located in regions or portionsof regions on SOST to which a BMP binds, such as peptides at the aminoterminal end of SOST and peptides that include amino terminal amino acidresidues and a portion of the core region (docking core) of SOST (e.g.,SEQ ID NOs:2-19, 21-28, and 47-50). The methods of the present inventionmay also be used to identify an antibody that impairs, prevents, orinhibits, formation of SOST homodimers. Such an antibody that bindsspecifically to SOST may be identified by detecting binding of theantibody to peptides that are derived from the core or the carboxyterminal region of SOST (e.g., SEQ ID NOs: 29-46 and 51-54).

Within other embodiments of the invention, methods are provided fordetermining whether an agent is capable of increasing bone mineralcontent, comprising the steps of (a) contacting a candidate agent tocells that express a TGF-beta binding-protein and (b) determiningwhether the expression of the TGF-beta binding-protein in the exposedcells decreases or whether an activity of the TGF-beta binding proteindecreases, and thereby determining whether the compound is capable ofincreasing bone mineral content. Within one embodiment, the cells mayinclude spontaneously transformed or untransformed normal human bonefrom bone biopsies or rat parietal bone osteoblasts.

Immunoassays may be used for detecting and quantifying the expression ofa TGF-beta binding protein and include, for example, CountercurrentImmuno-Electrophoresis (CIEP), radioimmunoassays,radioimmunoprecipitations, Enzyme-Linked Immuno-Sorbent Assays (ELISA),immunoblot assays such as dot blot assays and Western blots, inhibitionor competition assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110and 4,486,530; see also Antibodies: A Laboratory Manual, supra). Suchimmunoassays may use an antibody that is specific for a TGF-beta bindingprotein such as the anti-sclerostin antibodies described herein, or mayuse an antibody that is specific for a reporter molecule that isattached to the TGF-beta binding protein. The level of polypeptideexpression may also be determined by quantifying the amount of TGF-betabinding protein that binds to a TGF-beta binding protein ligand. By wayof example, binding of sclerostin in a sample to a BMP may be detectedby surface plasmon resonance. Alternatively, the level of expression ofmRNA encoding the specific TGF-beta binding protein may be quantified.

In other embodiments, an antibody specific for SOST may be used in amethod, such as a competition assay, in which candidate agents arescreened to identify an agent that will compete with the antibody forbinding to the TGF-binding protein. The interaction between an antibodyand a TGF-beta protein may be determined using immunoassay methods knownin the art and described herein.

By way of example, a family member of the TGF-beta superfamily, such asa TGF-beta binding protein or a TGF-beta superfamily member that is aligand of TGF-beta binding protein, is first bound to a solid phase,followed by addition of a candidate agent. A ligand of the TGF-betasuperfamily member that is bound to the solid phase is addedconcurrently or added in a subsequent step, the solid phase washed, andthe quantity of ligand that bound to the family member determined, whichindicates whether the candidate agent blocks binding of the ligand tothe TGF-beta member. Alternatively, the ligand of the TGF-betasuperfamily member may be attached to the solid phase, and the TGF-betasuperfamily member may be added concurrently or subsequently to theaddition of the candidate molecule. The amount of ligand that binds tothe TGF-beta superfamily member can be measured by labeling the ligandwith a detectable molecule according to methods known in the art anddescribed herein. Alternatively, the binding of the ligand to theTGF-beta superfamily member may be measured by adding one or moredetecting molecules, such as an antibody or fragment thereof thatspecifically binds to the ligand and quantifying the amount of themolecule that binds to the ligand. Agents that are suitable for use inincreasing bone mineral content as described herein are those agentsthat decrease the binding of TGF-beta binding protein to its ligand thatis a member of the TGF-beta superfamily in a statistically significantmanner.

Within other embodiments of the invention, methods are provided fordetermining whether an agent is capable of increasing bone mineralcontent, comprising the steps of (a) contacting a candidate agent tocells that express a TGF-beta protein and (b) determining whether theactivity of TGF-beta protein from the exposed cells is altered, andtherefrom determining whether the compound is capable of increasing bonemineral content. Similar to the methods described herein, a wide varietyof methods may be used to assess changes in TGF-beta binding-proteinexpression when a cell producing the TGF-beta binding protein is exposedto a selected test compound.

Within another embodiment of the present invention, methods are providedfor determining whether an agent is capable of increasing bone mineralcontent, comprising the steps of (a) contacting a candidate agent with aTGF-beta-binding-protein and a selected member of the TGF-beta family ofproteins, and (b) determining whether the selected agent up-regulatesthe signaling of the TGF-beta family of proteins or inhibits the bindingof the TGF-beta binding-protein to the TGF-beta family member. Withincertain embodiments, the molecule enhances the ability of a TGF-betafamily member to function as a positive regulator of mesenchymal celldifferentiation.

Similar to the above-described methods, a variety of methods may be usedto assess stimulation of a TGF-beta family member by a test compound,such as an antibody that specifically binds to SOST. One suchrepresentative method (see also Durham et al., Endo. 136:1374-1380)comprises attaching a TGF-beta family member, such as a BMP (e.g.,BMP-2, BMP-4, BMP-5, BMP-6, BMP-7) to the solid phase at a concentrationthat is equivalent to its Kd. The dissociation constant (Kd) may bedetermined according to methods for measuring binding rate constantsknown in the art, such as surface plasmon resonance using a BIAcoreinstrument according to techniques known in the art and described by themanufacturer (Biosensor, Piscataway, N.J.). A collection of antagonistcandidate agents is then added at a fixed concentration (typically, 20μM for small organic molecule collections (an isolated organic smallmolecule as used herein means that the molecule is greater than 90% pureas determined according to methods well known in the art (e.g., NMR,melting point) and 1 μM for candidate antibodies). An agent isconsidered an antagonist of this interaction when the agent inhibitsbinding of the BMP to SOST, that is, a decrease in the level of bindingis observed, by at least 40%, preferably 50% or 60%, and more preferably80% or 90% compared to the level of BMP binding to SOST that is observedin the absence of the agent. Such an agent may be further evaluated as apotential inhibitor on the basis of titration studies according to whichits inhibition constant and its influence on TGF-beta binding-proteinbinding affinity is determined. Comparable specificity control assaysmay also be conducted to establish the selectivity profile for theidentified antagonist according to studies using assays dependent on theBMP ligand action (e.g. BMP/BMP receptor competition study).

Within yet other embodiments of the present invention, methods areprovided for determining whether a candidate agent is capable ofincreasing bone mineral content, comprising the step of determiningwhether a candidate agent inhibits the binding of TGF-betabinding-protein to bone, or an analogue thereof. As used herein, itshould be understood that bone or analogues thereof refers tohydroxyapatite, or a surface composed of a powdered form of bone,crushed bone or intact bone. Similar to the methods described herein, awide variety of methods may be used to assess the inhibition of TGF-betabinding-protein localization to bone matrix (see, e.g., Nicolas et al.,Calcif Tissue Int. 47:206-12 (1995)).

While the methods described herein may refer to the analysis of anindividual candidate agent, the present invention should not be solimited. In particular, the agent may be contained within a mixture ofcandidate agents. As described herein, the candidate agents may be smallorganic molecules or may be antibodies or fragments thereof or otherpeptides or polypeptides. Antibodies or fragments may be identified byscreening a library of antibodies or antibody fragments or prepared bymethods described herein. Hence, the recited methods may furthercomprise the step of isolating an agent that inhibits the binding ofTGF-beta binding-protein to a TGF-beta family member.

In one embodiment of the invention, an antibody or antigen-bindingfragment thereof that specifically binds to a SOST polypeptide iscapable of competitively inhibiting binding of a TGF-beta family memberto the SOST polypeptide. The capability of the antibody or antibodyfragment to impair or blocking binding of a TGF-beta family member, suchas a BMP, to SOST may be determined according to any of the methodsdescribed herein. The antibody or fragment thereof that specificallybinds to SOST may impair, block, or prevent binding of a TGF-beta familymember to SOST by impairing SOST homodimer formation. An antibody thatspecifically binds to SOST may also be used to identify an activity ofSOST by inhibiting or impairing SOST from binding to a BMP.Alternatively, the antibody or fragment thereof may be incorporated in acell-based assay or in an animal model in which SOST has a definedactivity to determine whether the antibody alters (increases ordecreases in a statistically significant manner) that activity. Anantibody or fragment thereof that specifically binds to SOST may be usedto examine the effect of such an antibody in a signal transductionpathway and thereby modulate (stimulate or inhibit) the signalingpathway. Preferably, binding of an antibody to SOST results in astimulation or induction of a signaling pathway.

Production of Proteins

Polypeptides described herein include the TGF binding protein sclerostinand variants thereof and antibodies or fragments thereof thatspecifically bind to sclerostin. The polynucleotides that encode thesepolypeptides include derivatives that are substantially similar to thepolynucleotides (and, when appropriate, to the proteins includingpeptides and polypeptides that are encoded by the polynucleotides andtheir derivatives). As used herein, a nucleotide sequence is deemed tobe “substantially similar” if (a) the nucleotide sequence is derivedfrom the coding region of the polynucleotides described herein andincludes, for example, portions of the sequence or allelic variations ofthe sequences discussed above, or alternatively, encodes a molecule thatinhibits the binding of TGF-beta binding-protein to a member of theTGF-beta family; (b) the polynucleotide is capable of hybridizing topolynucleotide as provided herein under moderate, high or very highstringency (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3nd ed., Cold Spring Harbor Laboratory Press, NY, 2001); and/or (c) thepolynucleotide sequences are degenerate with respect to codon sequencesfor a particular amino acid of the polynucleotide sequences described in(a) or (b). Further, the nucleic acid molecule disclosed herein includesboth complementary and non-complementary sequences, provided thesequences otherwise meet the criteria set forth herein. Within thecontext of the present invention, high stringency means standardhybridization conditions (e.g., 5×SSPE, 0.5% SDS at 55-65° C., or theequivalent; 5×SSPE (1×SSPE=180 mM sodium chloride; 10 mM sodiumphosphate; 1 mM EDTA (pH 7.7); 5×Denhardt's solution (100×Denhardt's=2%(w/v) bovine serum albumin, 2% (w/v) Ficoll, 2% (w/v)polyvinylpyrrolidone); and 0.5% SDS). Post-hybridization washes at highstringency are typically performed in 0.5×SSC (1×SSC=150 mM sodiumchloride, 15 mM trisodium citrate) or in 0.5×SSPE at 55-60° C.).

Polynucleotides encoding SOST may be isolated from genomic or cDNAlibraries, from cells in a biological sample, tissues, or cell lines,and prepared using any variety of techniques (see, e.g., Sambrook,supra). For example, a polynucleotide may be amplified from cDNAprepared from a suitable cell or tissue. These polynucleotides may beamplified by polymerase chain reaction (PCR) using sequence specificprimers that are designed on the basis of the sequences provided herein,and which may be purchased or synthesized. Polynucleotides may also beobtained by screening cDNA or genomic DNA libraries using sequencespecific probes and primers. General methods for screening libraries byPCR are provided by, for example, Yu et al., “Use of the PolymeraseChain Reaction to Screen Phage Libraries,” in Methods in MolecularBiology, Vol. 15: PCR Protocols: Current Methods and Applications, White(ed.), pages 211-215 (Humana Press, Inc. 1993). Moreover, techniques forusing PCR to isolate related genes are described by, for example,Preston, “Use of Degenerate Oligonucleotide Primers and the PolymeraseChain Reaction to Clone Gene Family Members,” in Methods in MolecularBiology, Vol. 15: PCR Protocols: Current Methods and Applications, White(ed.), pages 317-337 (Humana Press, Inc. 1993).

The sequence of a TGF-beta binding-protein cDNA or TGF-betabinding-protein genomic fragment can be determined using standardmethods. Moreover, the identification of genomic fragments containing aTGF-beta binding-protein promoter or regulatory element can be achievedusing well-established techniques, such as deletion analysis (see,generally, Ausubel (1995)).

As an alternative, a polynucleotide encoding a TGF-beta binding-proteincan be obtained by synthesizing DNA molecules using mutually priminglong oligonucleotides and the nucleotide sequences described herein(see, e.g., Ausubel (1995) at pages 8-8 to 8-9). If PCR is incorporatedinto the method DNA molecules at least two kilobases in length can besynthesized (Adang et al., Plant Molec. Biol. 21:1131, 1993; Bambot etal., PCR Methods and Applications 2:266, 1993; Dillon et al., “Use ofthe Polymerase Chain Reaction for the Rapid Construction of SyntheticGenes,” in Methods in Molecular Biology, Vol. 15: PCR Protocols: CurrentMethods and Applications, White (ed.), pages 263-268, (Humana Press,Inc. 1993); Holowachuk et al., PCR Methods Appl. 4:299, 1995).

Nucleic acid molecules encoding a variant TGF-beta binding-protein canbe obtained by screening various cDNA or genomic libraries withpolynucleotide probes having nucleotide sequences based upon SEQ IDNO:55-57, 59, 61, 63, 65, 67, and 69, using procedures described herein.TGF-beta binding-protein polynucleotide variants can also be constructedsynthetically. For example, a nucleic acid molecule can be designed thatencodes a polypeptide having a conservative amino acid change whencompared with the amino acid sequence of SEQ ID NOs: 1, 20, 58, 60, 62,64, 66, 68, or 70. That is, variants can be obtained that contain one ormore amino acid substitutions of SEQ ID NOs: 1, 20, 58, 60, 62, 64, 66,68, or 70, in which an alkyl amino acid is substituted for an alkylamino acid in a TGF-beta binding-protein amino acid sequence; anaromatic amino acid is substituted for an aromatic amino acid in aTGF-beta binding-protein amino acid sequence; a sulfur-containing aminoacid is substituted for a sulfur-containing amino acid in a TGF-betabinding-protein amino acid sequence; a hydroxy-containing amino acid issubstituted for a hydroxy-containing amino acid in a TGF-betabinding-protein amino acid sequence; an acidic amino acid is substitutedfor an acidic amino acid in a TGF-beta binding-protein amino acidsequence; a basic amino acid is substituted for a basic amino acid in aTGF-beta binding-protein amino acid sequence; or a dibasicmonocarboxylic amino acid is substituted for a dibasic monocarboxylicamino acid in a TGF-beta binding-protein amino acid sequence.

Among the common amino acids, for example, a conservative amino acidsubstitution is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine (non-polar alkyl group-containing side chain); (2)phenylalanine, tyrosine, and tryptophan (aromatic side chain) (3) serineand threonine (hydroxyl group side chain), (4) aspartate and glutamate(carboxylic acid group side chain); (5) glutamine and asparagines (amidegroup-containing side chain) and (6) lysine, arginine and histidine(amino group side chain). In making such substitutions, it is importantto, where possible, maintain the cysteine backbone outlined in FIG. 1.

Conservative amino acid changes in SOST can be introduced bysubstituting nucleotides for the nucleotides recited in any one of SEQID NOs: 55-57, 59, 61, 63, 65, 67, or 69. Such conservative amino acidvariants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like (see Ausubel (1995) at pages8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A PracticalApproach (IRL Press 1991)). The functional activity of such variants canbe determined using standard methods and assays described herein.Alternatively, a variant TGF-beta binding-protein polypeptide retainsthe ability to specifically bind to anti-SOST antibodies.

Routine deletion analyses of nucleic acid molecules can be performed toobtain “functional fragments” of a nucleic acid molecule that encodes aTGF-beta binding-protein polypeptide. By way of illustration, DNAmolecules having the nucleotide sequence of any one of SEQ ID NOs:55-57,59, 61, 63, 65, 67, or 69 can be digested with Bal31 nuclease to obtaina series of nested deletions. The fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for activity, or for the ability tobind anti-TGF-beta binding-protein antibodies. One alternative toexonuclease digestion is to use oligonucleotide-directed mutagenesis tointroduce deletions or stop codons to specify production of a desiredfragment. Alternatively, particular fragments of a TGF-betabinding-protein encoding polynucleotide can be synthesized using thepolymerase chain reaction. In addition to assays and methods disclosedherein, standard techniques for functional analysis of proteins aredescribed by, for example, Treuter et al., Molec. Gen. Genet. 240:113,1993; Content et al., “Expression and preliminary deletion analysis ofthe 42 kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J.Biol. Chem. 270:29270, 1995; Fukunaga et al., J. Biol. Chem. 270:25291,1995; Yamaguchi et al., Biochem. Pharmacol. 50:1295, 1995; and Meisel etal., Plant Molec. Biol. 30:1, 1996.

The structure of the polypeptides encoded by the nucleic acid moleculesdescribed herein may be predicted from the primary translation productsusing the hydrophobicity plot function of, for example, P/C Gene orIntelligenetics Suite (Intelligenetics, Mountain View, Calif.), oraccording to the methods described by Kyte and Doolittle (J. Mol. Biol.157:105-132, 1982). Polypeptides may be prepared in the form of acidicor basic salts or in neutral form. In addition, individual amino acidresidues may be modified by oxidation or reduction. Furthermore, varioussubstitutions, deletions, or additions may be made to the amino acid ornucleic acid sequences, the net effect of which is to retain or furtherenhance or decrease the biological activity of the mutant or wild-typeprotein. Preferably, a SOST variant, or a fragment thereof retains orhas enhanced ability to bind to a SOST specific antibody. Moreover, dueto degeneracy in the genetic code, for example, the nucleotide sequencesencoding the same amino acid sequence may vary considerably.

Other derivatives of the proteins provided herein include conjugates ofthe proteins with other proteins or polypeptides. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins that may be added to facilitate purification oridentification of proteins (see U.S. Pat. No. 4,851,341, see also, Hoppet al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteinssuch as FLAG®/TGF-beta binding-protein may be constructed in order toassist in the identification, expression, and analysis of the protein.

Proteins described herein may be constructed using a wide variety oftechniques described herein. Further, mutations may be introduced atparticular loci by synthesizing oligonucleotides that contain a mutantsequence that are flanked by restriction sites, enabling ligation tofragments of the native sequence. Following ligation, the resultingreconstructed sequence encodes a derivative having the desired aminoacid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredpolynucleotide having particular codons altered according to thesubstitution, deletion, or insertion. Exemplary methods of making thealterations set forth above are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods,Plenum Press, 1981); and Sambrook et al. (supra). Deletion or truncationderivatives of proteins (e.g., a soluble extracellular portion) may alsobe constructed by using convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al. (MolecularCloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor LaboratoryPress (2001)).

Mutations that are made in the nucleic acid molecules preferablypreserve the reading frame of the coding sequences. Furthermore, themutations will preferably not create complementary regions that whentranscribed could hybridize to produce secondary mRNA structures, suchas loops or hairpins, which would adversely affect translation of themRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed mutants screened for gain, loss, or retention of biologicalactivity. Alternatively, mutations may be introduced at particular lociby synthesizing oligonucleotides containing a mutant sequence, flankedby restriction sites enabling ligation to fragments of the nativesequence. Following ligation, the resulting reconstructed sequenceencodes a derivative having the desired amino acid insertion,substitution, or deletion. Nucleic acid molecules that encode proteinsof the present invention may also be constructed using techniques suchas polymerase chain reaction (PCR) mutagenesis, chemical mutagenesis(Drinkwater and Klinedinst, PNAS 83:3402-3406, 1986), forced nucleotidemisincorporation (e.g., Liao and Wise Gene 88:107-111, 1990), or use ofrandomly mutagenized oligonucleotides (Horwitz et al., Genome 3:112-117,1989).

The present invention also provides for the manipulation and expressionof the polynucleotides provided herein and of polynucleotides encodingthe subject invention antibodies by culturing host cells containing avector capable of expressing such polynucleotides. An expression vectorrefers to recombinant nucleic acid construct that is capable ofdirecting the expression of desired protein. The vector may be composedof deoxyribonucleic acids (DNA), synthetic or cDNA-derived, ribonucleicacids (RNA), or a combination of the two (e.g., a DNA-RNA chimera). AcDNA polynucleotide is generally understood to mean a DNA polynucleotideprepared by transcribing an RNA molecule, such as mRNA. These vectors orvector constructs that include a polynucleotide sequence encoding thedesired protein preferably are operably linked to suitabletranscriptional or translational regulatory elements. Suitableregulatory elements may be derived from a variety of sources, includingbacterial, fungal, viral, mammalian, insect, or plant genes. Selectionof appropriate regulatory elements is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of regulatory elements include a transcriptional promoter andenhancer or RNA polymerase binding sequence, a transcriptionalterminator, and a ribosomal binding sequence including a translationinitiation signal. Optionally, the vector may include a polyadenylationsequence, one or more restriction sites, as well as one or moreselectable markers such as neomycin phosphotransferase or hygromycinphosphotransferase or any other markers known in the art. Additionally,depending on the host cell chosen and the vector employed, other geneticelements such as an origin of replication, additional nucleic acidrestriction sites, enhancers, sequences conferring inducibility oftranscription, and selectable markers, may also be incorporated into thevectors described herein.

Nucleic acid molecules that encode any of the proteins described abovemay be readily expressed by a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,insect, or plant cells. Methods for transforming or transfecting suchcells to express foreign DNA are well known in the art (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl.Acad. Sci. USA 75:1929-1933, 1978; Murray et al., U.S. Pat. No.4,801,542; Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S.Pat. No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel etal., U.S. Pat. No. 4,766,075; and Sambrook et al. Molecular Cloning: ALaboratory Manual, 3nd ed., Cold Spring Harbor Laboratory Press, 2001;for plant cells see Czako and Marton, Plant Physiol. 104:1067-1071,1994; and Paszkowski et al., Biotech. 24:387-392, 1992).

Bacterial host cells suitable for carrying out the present inventioninclude E. coli BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I,DH5, DH5I, DH51F′, DH51MCR, DH10B, DH10B/p3, DH11S, C600, HB11, JM11,JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647(see, for example, Brown (Ed.), Molecular Biology Labfax (Academic Press1991)); B. subtilis (BR151, YB886, M1119, M1120, and B170 (see, forexample, Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A PracticalApproach, Glover (Ed.) (IRL Press 1985)); Salmonella typhimurium; andvarious species within the genera Pseudomonas, Streptomyces, andStaphylococcus, as well as many other bacterial species well known toone of ordinary skill in the art. A representative example of abacterial host cell includes E. coli DH50α (Stratagene, LaJolla,Calif.).

Bacterial expression vectors preferably comprise a promoter thatfunctions in the host cell, one or more selectable phenotypic markers,and a bacterial origin of replication. Representative promoters includethe β-lactamase (penicillinase) and lactose promoter system (see Changet al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studieret al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin etal., Gene 87:123-126, 1990), the trp promoter (Nichols and Yanofsky,Meth. in Enzymology 101:155, 1983), and the tac promoter (Russell etal., Gene 20:231, 1982). Additional promoters include promoters capableof recognizing the T4, T3, Sp6 and T7 polymerases, the P_(R) and P_(L)promoters of bacteriophage lambda, the recA, heat shock, lacUV5, tac,lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.subtilis, the promoters of the bacteriophages of Bacillus, Streptomycespromoters, the int promoter of bacteriophage lambda, the bla promoter ofpBR322, and the CAT promoter of the chloramphenicol acetyl transferasegene. Prokaryotic promoters have been reviewed by Glick, J. Ind.Microbiol. 1:277, 1987, Watson et al., Molecular Biology of the Gene,4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995).Representative selectable markers include various antibiotic resistancemarkers such as the kanamycin or ampicillin resistance genes. Manyplasmids suitable for transforming host cells are well known in the art,including among others, pBR322 (see Bolivar et al., Gene 2:95, 1977),the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth. inEnzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268,1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, LaJolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others, Saccharomyces pombe, Saccharomycescerevisiae, the genera Pichia or Kluyveromyces and various species ofthe genus Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349).Suitable expression vectors for yeast and fungi include, among others,YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3(Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl.Acad. Sci. USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene8:121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978)and derivatives thereof.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080, 1980;Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum,New York, 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983). Examples ofuseful promoters for fungi vectors include those derived fromAspergillus nidulans glycolytic genes, such as the adh3 promoter(McKnight et al., EMBO J. 4:2093-2099, 1985). The expression units mayalso include a transcriptional terminator. An example of a suitableterminator is the adh3 terminator (McKnight et al., supra, 1985).

As with bacterial vectors, the yeast vectors will generally include aselectable marker, which may be one of any number of genes that exhibita dominant phenotype for which a phenotypic assay exists to enabletransformants to be selected. Preferred selectable markers are thosethat complement host cell auxotrophy, provide antibiotic resistance, orenable a cell to utilize specific carbon sources, and include leu2(Broach et al., ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3(Struhl et al., ibid.). Another suitable selectable marker is the catgene, which confers chloramphenicol resistance on yeast cells. Manyyeast cloning vectors have been designed and are readily available.These vectors include YIp-based vectors such as YIp5, YRp vectors suchas YRp17, YEp vectors such as YEp13, and YCp vectors such as YCp19. Oneskilled in the art will appreciate that a wide variety of suitablevectors are available for expression in yeast cells.

Techniques for transforming fungi are well known in the literature andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978), Yelton et al. (Proc.Natl. Acad. Sci. USA 81:1740-1747, 1984), and Russell (Nature301:167-169, 1983). The genotype of the host cell may contain a geneticdefect that is complemented by the selectable marker present on theexpression vector. Choice of a particular host and selectable marker iswell within the level of ordinary skill in the art.

Protocols for the transformation of yeast are also well known to thoseof ordinary skill in the art. For example, transformation may be readilyaccomplished either by preparation of spheroplasts of yeast with DNA(see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment withalkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163,1983). Transformation of fungi may also be carried out usingpolyethylene glycol as described by Cullen et al. (Bio/Technology 5:369,1987).

Viral vectors include those that comprise a promoter that directs theexpression of an isolated nucleic acid molecule encoding a desiredprotein as described above. A wide variety of promoters may be usedwithin the context of the present invention, including for example,promoters such as MoMLV LTR; RSV LTR (e.g., Gorman et al., Proc. Natl.Acad. Sci. USA 79:6777, 1982); Friend MuLV LTR; adenoviral promoter(Ohno et al., Science 265:781-784, 1994); neomycin phosphotransferasepromoter/enhancer; late parvovirus promoter (Koering et al., Hum. GeneTherap. 5:457-463, 1994); Herpes TK promoter (see, e.g., McKnight, Cell31:355, 1982); SV40 promoter (e.g., SV40 early promoter (Benoist et al.,Nature 290:304, 1981); metallothionein IIa gene enhancer/promoter; mousemetallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273,1982); mouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)); cytomegalovirus immediateearly promoter, and the cytomegalovirus immediate late promoter.Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control TGF-beta binding-proteingene expression in mammalian cells if the prokaryotic promoter isregulated by a eukaryotic promoter (Zhou et al., Mol. Cell. Biol.10:4529, 1990; Kaufman et al., Nuc. Acids Res. 19:4485, 1991).

Within particularly preferred embodiments of the invention, the promoteris a tissue-specific promoter (see e.g., WO 91/02805; EP 0,415,731; andWO 90/07936). Representative examples of suitable tissue specificpromoters include neural specific enolase promoter, platelet derivedgrowth factor beta promoter, bone morphogenic protein promoter, humanalpha1-chimaerin promoter, synapsin I promoter and synapsin II promoter.In addition to the above-noted promoters, other viral-specific promoters(e.g., retroviral promoters (including those noted above, as well asothers such as HIV promoters), hepatitis, herpes (e.g., EBV), andbacterial, fungal or parasitic (e.g., malarial) -specific promoters maybe utilized in order to target a specific cell or tissue that isinfected with a virus, bacteria, fungus, or parasite.

Mammalian cells suitable for carrying out the present invention include,among others COS, CHO, SaOS, osteosarcomas, KS483, MG-63, primaryosteoblasts, and human or mammalian bone marrow stroma. Additionalmammalian host cells include African green monkey kidney cells (Vero;ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573),baby hamster kidney cells (BHK-21; ATCC CRL 8544), canine kidney cells(MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61),rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rathepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidneycells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCCCRL 1658).

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Bone specific promoters include the promoter forbone sialo-protein and the promoter for osteocalcin. Viral promotersinclude the cytomegalovirus immediate early promoter (Boshart et al.,Cell 41:521-530, 1985), cytomegalovirus immediate late promoter, SV40promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981), MMTV LTR,RSV LTR, metallothionein-1, adenovirus E1a. Cellular promoters includethe mouse metallothionein-1 promoter (Palmiter et al., U.S. Pat. No.4,579,821), a mouse V_(κ) promoter (Bergman et al., Proc. Natl. Acad.Sci. USA 81:7041-7045, 1983; Grant et al., Nucleic Acids Res. 15:5496,1987) and a mouse V_(H) promoter (Loh et al., Cell 33:85-93, 1983). Thechoice of promoter will depend, at least in part, upon the level ofexpression desired or the recipient cell line to be transfected.

Such expression vectors may also contain a set of RNA splice siteslocated downstream from the promoter and upstream from the DNA sequenceencoding the peptide or protein of interest. Preferred RNA splice sitesmay be obtained from adenovirus and/or immunoglobulin genes. Alsocontained in the expression vectors is a polyadenylation signal locateddownstream of the coding sequence of interest. Suitable polyadenylationsignals include the early or late polyadenylation signals from SV40(Kaufman and Sharp, ibid.), the polyadenylation signal from theAdenovirus 5 E1B region and the human growth hormone gene terminator(DeNoto et al., Nucleic Acids Res. 9:3719-3730, 1981). The expressionvectors may include a noncoding viral leader sequence, such as theAdenovirus 2 tripartite leader, located between the promoter and the RNAsplice sites. Preferred vectors may also include enhancer sequences,such as the SV40 enhancer. Expression vectors may also include sequencesencoding the adenovirus VA RNAs. Suitable expression vectors can beobtained from commercial sources (e.g., Stratagene, La Jolla, Calif.).

Vector constructs comprising cloned DNA sequences can be introduced intocultured mammalian cells by, for example, liposome-mediatedtransfection, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), or DEAE-dextran mediatedtransfection (Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley and Sons, Inc., NY, 1987); retroviral, adenoviraland protoplast fusion-mediated transfection (see Sambrook et al.,supra). Naked vector constructs can also be taken up by muscular cellsor other suitable cells subsequent to injection into the muscle of amammal (or other animal). To identify cells that have been stablytransfected with the vector containing the cloned DNA, a selectablemarker is generally introduced into the cells along with the gene orcDNA of interest. Preferred selectable markers for use in culturedmammalian cells include genes that confer resistance to drugs, such asneomycin, hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. Preferred amplifiable selectable markersare the DHFR gene and the neomycin resistance gene. Selectable markersare reviewed by Thilly (Mammalian Cell Technology, ButterworthPublishers, Stoneham, Mass.).

Mammalian cells containing a suitable vector are allowed to grow for aperiod of time, typically 1-2 days, to begin expressing the DNAsequence(s) of interest. Drug selection is then applied to select forgrowth of cells that are expressing the selectable marker in a stablefashion. For cells that have been transfected with an amplifiable,selectable marker, the drug concentration may be increased in a stepwisemanner to select for increased copy number of the cloned sequences,thereby increasing expression levels. Cells expressing the introducedsequences are selected and screened for production of the protein ofinterest in the desired form or at the desired level. Cells that satisfythese criteria can then be cloned and scaled up for production.

Numerous insect host cells known in the art can also be useful withinthe present invention. For example, baculoviruses as vectors forexpressing heterologous DNA sequences in insect cells may be used(Atkinson et al., Pestic. Sci. 28:215-224 (1990)). The baculovirussystem provides an efficient means to introduce cloned TGF-betabinding-protein encoding polynucleotides into insect cells. Suitableexpression vectors are based upon the Autographa californica multiplenuclear polyhedrosis virus (AcMNPV), which contain well known promoterssuch as Drosophila heat shock protein (hsp) 70 promoter, Autographacalifornica nuclear polyhedrosis virus immediate-early gene promoter(ie-1), and the delayed early 39K promoter, baculovirus p10 promoter,and the Drosophila metallothionein promoter. Suitable insect host cellsinclude cell lines derived from IPLB-Sf-21, a Spodoptera frugiperdapupal ovarian cell line, such as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21(Invitrogen Corporation; San Diego, Calif.), as well as DrosophilaSchneider-2 cells. Established techniques for producing recombinantproteins in baculovirus systems are provided by Bailey et al.,“Manipulation of Baculovirus Vectors,” in Methods in Molecular Biology,Volume 7: Gene Transfer and Expression Protocols, Murray (ed.), pages147-168 (The Humana Press, Inc. 1991); Patel et al., “The baculovirusexpression system,” in DNA Cloning 2: Expression Systems, 2nd Edition,Glover et al. (eds.), pages 205-244 (Oxford University Press 1995);Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995); and Lucknow, “InsectCell Expression Technology,” in Protein Engineering Principles andPractice, Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.1996).

Alternatively, numerous plant host cells are known in the art and canalso be useful for expressing polynucleotides, for example,Agrobacterium rhizogenes (Sinkar et al. J. Biosci. (Bangalore) 11:47-58,1987). Expression vectors can also be introduced into plant protoplasts,intact plant tissues, or isolated plant cells. General methods ofculturing plant tissues are provided, for example, by Miki et al.,“Procedures for Introducing Foreign DNA into Plants,” in Methods inPlant Molecular Biology and Biotechnology, Glick et al. (eds.), pages67-88 (CRC Press, 1993).

Within related embodiments of the present invention, proteins of thepresent invention may be expressed in a transgenic animal whose germcells and somatic cells contain a gene that encodes the desired proteinand that is operatively linked to a promoter effective for theexpression of the gene. Alternatively, in a similar manner, transgenicanimals may be prepared that lack the desired gene (e.g., “knock-out”mice). Such transgenics may be prepared in a variety of non-humananimals, including mice, rats, rabbits, sheep, dogs, goats, and pigs(see Hammer et al., Nature 315:680-683, 1985, Palmiter et al., Science222:809-814, 1983, Brinster et al., Proc. Natl. Acad. Sci. USA82:4438-4442, 1985, Palmiter and Brinster, Cell 41:343-345, 1985, andU.S. Pat. Nos. 5,175,383, 5,087,571, 4,736,866, 5,387,742, 5,347,075,5,221,778, and 5,175,384). Briefly, an expression vector, including anucleic acid molecule to be expressed together with appropriatelypositioned expression control sequences, is introduced into pronuclei offertilized eggs, for example, by microinjection. Integration of theinjected DNA is detected by blot analysis of DNA from tissue samples. Itis preferred that the introduced DNA be incorporated into the germ lineof the animal so that it is passed on to the animal's progeny.Tissue-specific expression may be achieved through the use of atissue-specific promoter, or through the use of an inducible promoter,such as the metallothionein gene promoter (Palmiter et al., 1983,supra), which allows regulated expression of the transgene.

Proteins can be isolated by, among other methods, culturing suitablehost and vector systems to produce the recombinant translation productsof the present invention. Supernatants from such cell lines, or proteininclusion bodies, or whole cells from which the protein is not secretedinto the supernatant, can then be treated by a variety of purificationprocedures in order to isolate the desired proteins. For example, thesupernatant may be first concentrated using commercially availableprotein concentration filters, such as an Amicon or Millipore Pelliconultrafiltration unit. Following concentration, the concentrate may beapplied to a suitable purification matrix such as an affinity matrix,for example, a specific antibody bound to a suitable support.Alternatively, anion or cation exchange resins or size-exclusionmatrices may be employed in order to purify the protein. As a furtheralternative, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps may be used to further purify theprotein. Other methods of isolating the proteins of the presentinvention are well known in the art. The purity of an isolated proteinor polypeptide may be determined by SDS-PAGE analysis followed byCoomassie blue staining or by silver staining.

General methods for expressing and recovering foreign protein producedby a mammalian cell system is provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc., 1995).

More generally, a TGF-beta binding-protein can be isolated by standardtechniques, such as affinity chromatography, size exclusionchromatography, ion exchange chromatography, HPLC, and the like.Additional variations in TGF-beta binding-protein isolation andpurification can be devised by those of skill in the art. For example,anti-TGF-beta binding-protein antibodies, obtained as described herein,can be used to isolate large quantities of protein by immunoaffinitypurification. Purification of antibodies of the present invention arefurther described herein.

Detectable Labels

A detectable label is a molecule or atom that can be conjugated to apolypeptide (including an antibody or fragment thereof) or apolynucleotide to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, enzymes, and other markermoieties. A TGF binding protein or an antibody that specifically bindsto the TGF binding protein or candidate molecules described above may belabeled with a variety of compounds including, for example, fluorescentmolecules, toxins, and radionuclides. Representative examples offluorescent molecules include fluorescein, Phycobili proteins, such asphycoerythrin, rhodamine, Texas red, and luciferase. Representativeexamples of toxins include ricin, abrin diphtheria toxin, cholera toxin,gelonin, pokeweed antiviral protein, tritin, Shigella toxin, andPseudomonas exotoxin A. Representative examples of radionuclides includeCu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111,I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211,Pb-212 and Bi-212. In addition, the antibodies described above may alsobe labeled or conjugated to one partner of a ligand binding pair.Representative examples include avidin-biotin, streptavidin-biotin, andriboflavin-riboflavin binding protein.

Methods for conjugating or labeling the molecules described herein withthe representative labels set forth above may be readily accomplished byone of ordinary skill in the art (see Trichothecene Antibody Conjugate,U.S. Pat. No. 4,744,981; Antibody Conjugate, U.S. Pat. No. 5,106,951;Fluorogenic Materials and Labeling Techniques, U.S. Pat. No. 4,018,884;Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Pat.No. 4,897,255; and Metal Radionuclide Chelating Compounds for ImprovedChelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Methods InEnzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B,Jakoby and Wilchek (eds.), Academic Press, New York, p. 30, 1974; seealso Wilchek and Bayer, “The Avidin-Biotin Complex in BioanalyticalApplications,” Anal. Biochem. 171:1-32, 1988). An immunoconjugate is acomposition comprising an antibody, such as an anti-TGF-beta bindingprotein antibody, or an antibody fragment thereof, and a detectablelabel. Preferably the antibody moiety of the immunoconjugatespecifically binds to its cognate antigen with the same or slightlyreduced binding affinity after conjugation as before conjugation.

Pharmaceutical Compositions

As noted above, the present invention also provides a variety ofpharmaceutical compositions, comprising one of the above-describedmolecules that inhibits binding of the TGF-beta binding-protein to amember of the TGF-beta family along with a pharmaceutically orphysiologically acceptable carrier, excipient, or diluent. Any suitablecarrier known to those of ordinary skill in the art may be employed inthe pharmaceutical compositions of the present invention. Carriers fortherapeutic use are well known, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroed. (1985)). Generally, such carriers should be nontoxic to recipientsat the dosages and concentrations employed. Ordinarily, the preparationof such compositions entails combining the therapeutic agent withbuffers; antioxidants such as ascorbic acid, low molecular weight (lessthan about 10 residues) polypeptides; proteins; amino acids;carbohydrates including maltose, glucose, sucrose, or dextrins;chelating agents such as EDTA; glutathione; and other stabilizers andexcipients. Neutral buffered saline or saline mixed with nonspecificserum albumin are exemplary diluents. Alternatively, compositions of thepresent invention may be formulated as a lyophilizate.

In general, the type of carrier is selected based on the mode ofadministration. Pharmaceutical compositions may be formulated for anyappropriate manner of administration, including, for example, topical,oral, nasal, intrathecal, rectal, vaginal, sublingual or parenteraladministration, including subcutaneous, intravenous, intramuscular,intrasternal, intracavernous, intrameatal, or intraurethral injection orinfusion. A pharmaceutical composition (e.g., for oral administration ordelivery by injection) may be in the form of a liquid (e.g., an elixir,syrup, solution, emulsion or suspension). A liquid pharmaceuticalcomposition may include, for example, one or more of the following:sterile diluents such as water for injection, saline solution,preferably physiological saline, Ringer's solution, isotonic sodiumchloride, fixed oils that may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents; antioxidants; chelating agents; buffers such asacetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. A parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. The use of physiological saline is preferred,and an injectable pharmaceutical composition is preferably sterile.

The compositions described herein may be formulated for sustainedrelease (i.e., a formulation such as a capsule or sponge that effects aslow release of compound following administration). Such compositionsmay generally be prepared using well known technology and administeredby, for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Sustained-release formulationsmay contain an agent dispersed in a carrier matrix and/or containedwithin a reservoir surrounded by a rate controlling membrane. Carriersfor use within such formulations are biocompatible, and may also bebiodegradable; preferably the formulation provides a relatively constantlevel of active component release. The amount of active compoundcontained within a sustained release formulation depends upon the siteof implantation, the rate and expected duration of release and thenature of the condition to be treated or prevented. Illustrativecarriers useful in this regard include microparticles ofpoly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose,dextran and the like. Other illustrative delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638).

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems, such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

In another illustrative embodiment, calcium phosphate core particles areemployed as carriers or as controlled release matrices for thecompositions of this invention. Exemplary calcium phosphate particlesare disclosed, for example, in published patent application No.WO/0046147.

For pharmaceutical compositions comprising a polynucleotide encoding ananti-SOST antibody and/or modulating agent (such that the polypeptideand/or modulating agent is generated in situ), the polynucleotide may bepresent within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid, and bacterial, viraland mammalian expression systems. Techniques for incorporating DNA intosuch expression systems are well known to those of ordinary skill in theart. The DNA may also be “naked,” as described, for example, in Ulmer etal., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Remington's PharmaceuticalSciences, 15th ed., pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. Moreover, for human administration, preparations will ofcourse preferably meet sterility, pyrogenicity, and the general safetyand purity standards as required by FDA Office of Biologics standards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol. 16(7):307-21, 1998;Takakura, Nippon Rinsho 56(3):691-95, 1998; Chandran et al., Indian J.Exp. Biol. 35(8):801-09, 1997; Margalit, Crit. Rev. Ther. Drug CarrierSyst. 12(2-3):233-61, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No.5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety). Liposomes have been used successfully with a number ofcell types that are normally difficult to transfect by other procedures,including T cell suspensions, primary hepatocyte cultures and PC 12cells (Renneisen et al., J. Biol. Chem. 265(27):16337-42, 1990; Mulleret al., DNA Cell Biol. 9(3):221-29, 1990). In addition, liposomes arefree of the DNA length constraints that are typical of viral-baseddelivery systems. Liposomes have been used effectively to introducegenes, various drugs, radiotherapeutic agents, enzymes, viruses,transcription factors, allosteric effectors and the like, into a varietyof cultured cell lines and animals. Furthermore, he use of liposomesdoes not appear to be associated with autoimmune responses orunacceptable toxicity after systemic delivery. In certain embodiments,liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev. Ind. Pharm. 24(12):1113-28, 1998). To avoid side effectsdue to intracellular polymeric overloading, such ultrafine particles(sized around 0.1 μm) may be designed using polymers able to be degradedin vivo. Such particles can be made as described, for example, byCouvreur et al., Crit. Rev. Ther. Drug Carrier Syst. 5(1):1-20, 1988;zur Muhlen et al., Eur. J. Pharm. Biopharm. 45(2):149-55, 1998; Zambauxet al., J. Controlled Release 50(1-3):31-40, 1998; and U.S. Pat. No.5,145,684.

In addition, pharmaceutical compositions of the present invention may beplaced within containers, along with packaging material that providesinstructions regarding the use of such pharmaceutical compositions.Generally, such instructions will include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) that may be necessary to reconstitute thepharmaceutical composition.

Methods of Treatment

The present invention also provides methods for increasing the mineralcontent and mineral density of bone. Briefly, numerous conditions resultin the loss of bone mineral content, including for example, disease,genetic predisposition, accidents that result in the lack of use of bone(e.g., due to fracture), therapeutics that effect bone resorption orthat kill bone forming cells, and normal aging. Through use of themolecules described herein that inhibit binding of the TGF-betabinding-protein to a TGF-beta family member, such conditions may betreated or prevented. As used herein, that bone mineral content isunderstood to have increased if bone mineral content has increased in astatistically significant manner at a selected site.

A wide variety of conditions that result in loss of bone mineral contentmay be treated with the molecules described herein. Patients with suchconditions may be identified through clinical diagnosis using well knowntechniques (see, e.g., Harrison's Principles of Internal Medicine,McGraw-Hill, Inc.). Representative examples of diseases that may betreated included dysplasias, wherein growth or development of bone isabnormal. Representative examples of such conditions includeachondroplasia, cleidocranial dysostosis, enchondromatosis, fibrousdysplasia, Gaucher's Disease, hypophosphatemic rickets, Marfan'ssyndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesisimperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions,fractures, periodontal disease, pseudoarthrosis, and pyogenicosteomyelitis.

Other conditions that may be treated or prevented include a wide varietyof causes of osteopenia (i.e., a condition that causes greater than onestandard deviation of bone mineral content or density below peakskeletal mineral content at youth). Representative examples of suchconditions include anemic states, conditions caused by steroids,conditions caused by heparin, bone marrow disorders, scurvy,malnutrition, calcium deficiency, idiopathic osteoporosis, congenitalosteopenia or osteoporosis, alcoholism, chronic liver disease, senility,postmenopausal state, oligomenorrhea, amenorrhea, pregnancy, diabetesmellitus, hyperthyroidism, Cushing's disease, acromegaly, hypogonadism,immobilization or disuse, reflex sympathetic dystrophy syndrome,transient regional osteoporosis, and osteomalacia.

Within one embodiment of the present invention, bone mineral content ordensity may be increased by administering to a warm-blooded animal atherapeutically effective amount of a molecule that inhibits a SOSTpolypeptide from binding to a TGF-beta family member. Examples ofwarm-blooded animals that may be treated include both vertebrates andmammals, including for example humans, horses, cows, pigs, sheep, dogs,cats, rats, and mice. Representative examples of therapeutic moleculesinclude ribozymes, ribozyme genes, antisense oligonucleotides, andantibodies as described herein.

Within other embodiments of the present invention, methods are providedfor increasing bone density comprising the steps of introducing intocells that home to bone, a vector that directs the expression of amolecule that inhibits binding of SOST to a member of the TGF-betafamily, and administering the vector containing cells to a warm-bloodedanimal. Briefly, cells that home to bone may be obtained directly fromthe bone of patients (e.g., cells obtained from the bone marrow such asCD34+, osteoblasts, osteocytes, and the like), from peripheral blood, orfrom cultures. Representative examples of suitable vectors include viralvectors such as herpes viral vectors (e.g., U.S. Pat. No. 5,288,641);adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Kolls et al., Proc.Natl. Acad. Sci. USA 91(1):215-219, 1994; Kass-Eisler et al., Proc.Natl. Acad. Sci. USA 90(24):11498-502, 1993; Guzman et al., Circulation88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993;Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum. Gene Ther.4(4):403-409, 1993; Caillaud et al., Eur. J. Neurosci. 5(10:1287-1291,1993; Vincent et al., Nat. Genet. 5(2):130-134, 1993; Jaffe et al., Nat.Genet. 1(5):372-378, 1992; and Levrero et al., Gene 101(2):195-202,1991); adeno-associated viral vectors (WO 95/13365; Flotte et al., PNAS90(22):10613-10617, 1993); baculovirus vectors; parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457-463, 1994); pox virus vectors(Panicali and Paoletti, Proc. Natl. Acad. Sci. USA 79:4927-4931, 1982;and Ozaki et al., Biochem. Biophys. Res. Comm. 193(2):653-660, 1993),and retroviruses (e.g., EP 0,415,731; WO 90/07936; WO 91/0285, WO94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO93/11230; WO 93/10218). Viral vectors may likewise be constructed thatcontain a mixture of different elements (e.g., promoters, envelopesequences, and the like) from different viruses or from non-viralsources. Within various embodiments, either the viral vector itself, ora viral particle that contains the viral vector may be used in themethods and compositions described below.

Within other embodiments of the invention, nucleic acid molecules thatencode a molecule that inhibits binding of a SOST polypeptide to amember of the TGF-beta family themselves may be administered by avariety of techniques including, for example, administration ofasialoosomucoid (ASOR) conjugated with poly-L-lysine DNA complexes(Cristano et al., PNAS 92122-92126, 1993); DNA linked to killedadenovirus (Curiel et al., Hum. Gene Ther. 3(2):147-154, 1992);cytofectin-mediated introduction (DMRIE-DOPE, Vical, Calif.); direct DNAinjection (Acsadi et al., Nature 352:815-818, 1991); DNA ligand (Wu etal., J. of Biol. Chem. 264:16985-16987, 1989); lipofection (Felgner etal., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989); liposomes(Pickering et al., Circ. 89(1):13-21, 1994; and Wang et al., PNAS84:7851-7855, 1987); microprojectile bombardment (Williams et al., Proc.Natl. Acad. Sci. USA 88:2726-2730, 1991); and direct delivery of nucleicacids which encode the protein itself either alone (Vile and Hart,Cancer Res. 53: 3860-3864, 1993), or utilizing PEG-nucleic acidcomplexes.

Determination of increased bone mineral content may be determineddirectly through the use of X-rays (e.g., Dual Energy X-rayAbsorptometry or “DEXA”), or by inference through bone turnover markerssuch as osteoblast specific alkaline phosphatase, osteocalcin, type 1procollagen C′ propeptide (PICP), and total alkaline phosphatase (seeComier, Curr. Opin. in Rheu. 7:243 (1995)), or by markers of boneresorption including, but not limited to, pyridinoline,deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasmatartrate-resistant acid phosphatases, and galactosyl hydroxylysine (seeComier, id.). The amount of bone mass may also be calculated from bodyweights or by using other methods (see Guinness-Hey, Metab. Bone Dis.Relat. Res. 5:177-181, 1984).

As will be evident to one of skill in the art, the amount and frequencyof administration will depend, of course, on such factors as the natureand severity of the indication being treated, the desired response, thecondition of the patient, and so forth. Typically, the compositions maybe administered by a variety of techniques, as noted above.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Modeling of Sclerostin Core Region

Homology recognition techniques (e.g., PSI-BLAST (Altschul et al.,Nucleic Acids Res. 25:3389-402 (1997)), FUGUE (Shi et al., J. Mol. Biol.310:243-57 (2001)) suggested that the core region of SOST (SOST_Core)adopts a cystine-knot fold. FUGUE is a sensitive method for detectinghomology between sequences and structures. Human Chorionic Gonadotropinβ (hCG-β), for which an experimentally determined 3D structure is known,was identified by FUGUE (Shi et al., supra) as the closest homologue ofSOST_Core. Therefore, hCG-β was used as the structural template to build3D models for SOST_Core.

An alignment of SOST_Core and its close homologues is shown in FIG. 1.Among the homologues shown in the alignment, only hCG-β (CGHB) had known3D structure. The sequence identity between SOST_Core and hCG-β wasapproximately 25%. Eight CYS residues were conserved throughout thefamily, emphasizing the overall structural similarity between SOST_Coreand hCG-β. Three pairs of cystines (1-5, 3-7, 4-8) formed disulfidebonds (shown with solid lines in FIG. 1) in a “knot” configuration,which is characteristic to the cystine-knot fold. An extra disulfidebond (2-6), shown as a dotted line in FIG. 1, was unique to this familyand distinguished the family of proteins from other cystine-knotfamilies (e.g., TGF-β, BMP).

SOST_Core was modeled using PDB (Berman et al., Acta Crystallogr. D.Biol. Cystallogr. 58(Pt 6 Pt1):899-907 (2002)) entry 1HCN, the 3Dstructure of hCG-β (Wu et al., Structure 2:545-58 (1994)), as thestructural template. Models were calculated with MODELER (Sali &Blundell, J. Mol. Biol. 234:779-815 (1993)). A snapshot of the bestmodel is shown in FIG. 2.

Most of the cystine-knot proteins form dimers because of the lack ofhydrophobic core in a monomer (Scheufler et al., supra; Schlunegger andGrutter, J. Mol. Biol. 231:445-58 (1993)); Wu et al., supra). SOSTlikely follows the same rule and forms a homodimer to increase itsstability. Constructing a model for the dimerized SOST_Core regionpresented several challenges because (1) the sequence similarity betweenSOST_Core and hCG-β was low (25%); (2) instead of a homodimer, hCG-βformed a heterodimer with hCG-α; and (3) a number of different relativeconformations of monomers have been observed in dimerized cystine-knotproteins from different families (e.g., PDGF, TGF-β, Neurotrophin,IL-17F, Gonadotropin), which suggested that the dimer conformation ofSOST could deviate significantly from the hCG-α/β heterodimerconformation. In constructing the model, hCG-α was replaced with hCG-βfrom the heterodimer structure (1HCN) using structure superimpositiontechniques combined with manual adjustment, and then a SOST_Corehomodimer model was built according to the pseudo hCG-β homodimerstructure. The final model is shown in FIG. 3.

Example 2 Modeling SOST-BMP Interaction

This example describes protein modeling of type I and type II receptorbinding sites on BMP that are involved with interaction between BMP andSOST.

Competition studies demonstrated that SOST competed with both type I andtype II receptors for binding to BMP. In an ELISA-based competitionassay, BMP-6 selectively interacted with the sclerostin-coated surface(300 ng/well) with high affinity (K_(D)=3.4 nM). Increasing amounts ofBMP receptor IA (FC fusion construct) competed with sclerostin forbinding to BMP-6 (11 nM) (IC₅₀=114 nM). A 10-fold molar excess of theBMP receptor was sufficient to reduce binding of sclerostin to BMP-6 byapproximately 50%. This competition was also observed with a BMPreceptor II-FC fusion protein (IC₅₀=36 nM) and DAN (IC₅₀=43 nM).Specificity of the assay was shown by lack of competition for binding toBMP-6 between sclerostin and a rActivin R1B-FC fusion protein, a TGF-βreceptor family member that did not bind BMP.

The type I and type II receptor binding sites on a BMP polypeptide havebeen mapped and were spatially separated (Scheufler et al., supra; Inniset al., supra; Nickel et al., supra; Hart et al. supra). Noggin, anotherBMP antagonist that binds to BMP with high affinity, contacts BMP atboth type I and type II receptor binding sites via the N-terminalportion of Noggin (Groppe et al., supra). The two β-strands in the coreregion near the C-terminal also contact BMP at the type II receptorbinding site.

A manually tuned alignment of Noggin and SOST indicated that the twopolypeptides shared sequence similarity between the N-terminal portionsof the proteins and between the core regions. An amino acid sequencealignment is presented in FIG. 4. The cysteine residues that form thecharacteristic cys-knot were conserved between Noggin and SOST. Theoverall sequence identity was 24%, and the sequence identity within theN-terminal binding region (alignment positions 1-45) was 33%. Tworesidues in the Noggin N-terminal binding region, namely Leu (L) atalignment position 21 and Glu (E) at position 23, were reported to playimportant roles in BMP binding (Groppe et al., supra). Both residueswere conserved in SOST as well. The sequence similarity within the coreregion (alignment positions 131-228) was about 20%, but the cys-knotscaffold was maintained and a sufficient number of key residues wasconserved, supporting the homology between Noggin and SOST.

The Noggin structure was compared to SOST also to understand how twoSOST monomers dimerize. As shown in FIG. 5, the Noggin structuresuggested that the linker between the N-terminal region and the coreregion not only played a role in connecting the two regions, but alsoformed part of the dimerization interface between two Noggin monomers.One major difference between Noggin and SOST was that the linker betweenthe N-terminal region and the core region was much shorter in SOST.

The C-terminal region of SOST may play a role in SOST dimerization. Thesequence of Noggin ended with the core region, while SOST had an extraC-terminal region. In the Noggin structure a disulfide bond connectedthe C-termini of two Noggin monomers. Thus, the C-terminal region ofSOST started close to the interface of two monomers and could contributeto dimerization. In addition, secondary structure prediction shows thatsome portions of the C-terminal region of SOST had a tendency to formhelices. This region in SOST may be responsible for the dimerizationactivity, possibly through helix-helix packing, which mimicked thefunction of the longer linker in Noggin. Another difference between thestructure of Noggin and SOST was the amino acid insertion in the SOSTcore region at alignment positions 169-185 (see FIG. 4). This insertionextended a β-hairpin, which pointed towards the dimerization interfacein the Noggin structure (shown in FIG. 5 as a loop region in the middleof the monomers and above the C-terminal Cys residue). This elongatedβ-hairpin could also contribute to SOST dimerization.

Example 3 Design and Preparation of SOST Peptide Immunogens

This Example describes the design of SOST peptide immunogens that areused for immunizing animals and generating antibodies that blockinteractions between BMP and SOST and prevent dimer formation of SOSTmonomers.

BMP Binding Fragments

The overall similarity between SOST and Noggin and the similaritybetween the N-terminal regions of the two polypeptides suggest that SOSTmay interact with BMP in a similar manner to Noggin. That is, theN-terminal region of SOST may interact with both the type I and type IIreceptor binding sites on BMP, and a portion of the core region (aminoacid alignment positions 190-220 in FIG. 4) may interact with the typeII receptor binding site such that antibodies specific for these SOSTregions may block or impair binding of BMP to SOST.

The amino acid sequences of these SOST polypeptide fragments for rat andhuman SOST are provided as follows.

SOST_N_Linker: The N-terminal region (includes the short linker thatconnects to the core region)

Human: (SEQ ID NO: 47) QGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYS Rat: (SEQ ID NO: 48)QGWQAFKNDATEIIPGLREYPEPPQELENNQTMNRAENGGRPPHHPYDT KDVSEYS

SOST_Core_Bind: Portion of the core region that is likely to contact BMPat its type II receptor binding site (extended slightly at both terminito include the CYS residue anchors):

Human: (SEQ ID NO: 49) CIPDRYRAQRVQLLCPGGEAPRARKVRLVASC Rat:(SEQ ID NO: 50) CIPDRYRAQRVQLLCPGGAAPRSRKVRLVASC

SOST Dimerization Fragments

The C-terminal region of SOST is likely to be involved in the formationof SOST homodimers (see Example 2). The elongated β-hairpin may alsoplay a role in homodimer formation. Antibodies that specifically bind tosuch regions may prevent or impair dimerization of SOST monomers, whichmay in turn interfere with interaction between SOST and BMP. Polypeptidefragments in rat and human SOST corresponding to these regions are asfollows.

SOST_C: the C-terminal Region

Human: (SEQ ID NO: 51) LTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY Rat:(SEQ ID NO: 52) LTRFHNQSELKDFGPETARPQKGRKPRPRARGAKANQAELENAY

SOST_Core_Dimer: Portion of the core region that is likely involved inSOST dimerization (extended slightly at both termini to include the Cysresidue anchors):

Human: CGPARLLPNAIGRGKWWRPSGPDFRC (SEQ ID NO: 53)Rat: CGPARLLPNAIGRVKWWRPNGPDFRC (SEQ ID NO: 54)

BMP Binding Fragment at SOST N-terminus

The key N-terminal binding region of SOST (alignment positions 1-35 inFIG. 4) was modeled on the basis of the Noggin/BMP-7 complex structure(Protein Data Bank Entry No: 1M4U) and the amino acid sequence alignment(see FIG. 4) to identify amino acid residues of the SOST N-terminus thatlikely interact with BMP. The model of SOST is presented in FIG. 6. Inthe comparative model, phenylalanine (Phe, F) at alignment position 8(see arrow and accompanying text) in the SOST sequence projects into ahydrophobic pocket on the surface of the BMP dimer. The same“knob-into-hole” feature has been observed in the BMP and type Ireceptor complex structure (Nickel et al., supra), where Phe85 of thereceptor fits into the same pocket, which is a key feature inligand-type I receptor recognition for TGF-β superfamily members(including, for example, TGF-β family, BMP family, and the like).According to the model, a proline (Pro) directed turn is also conserved,which allows the N-terminal binding fragment to thread along the BMPdimer surface, traveling from type I receptor binding site to type IIreceptor binding site on the other side of the complex. Also conservedis another Pro-directed turn near the carboxy end of the bindingfragment, which then connects to the linker region. Extensive contactsbetween SOST and BMP are evident in FIG. 6.

Peptide Immunogens

Peptides were designed to encompass the SOST N-terminal region predictedto make contact with BMP proteins. The peptide sequences are presentedbelow. For immunizing animals, the peptide sequences were designed tooverlap, and an additional cysteine was added to the C-terminal end tofacilitate crosslinking to KLH. The peptides were then used forimmunization. The peptide sequences of the immunogens are as follows.

Human SOST:

QGWQAFKNDATEIIPELGEY (SEQ ID NO: 2) TEIIPELGEYPEPPPELENN (SEQ ID NO: 3)PEPPPELENNKTMNRAENGG (SEQ ID NO: 4) KTMNRAENGGRPPHHPFETK (SEQ ID NO: 5)RPPHHPFETKDVSEYS (SEQ ID NO: 6)

Human SOST Peptides with Additional Cys:

QGWQAFKNDATEIIPELGEY-C (SEQ ID NO: 7) TEIIPELGEYPEPPPELENN-C(SEQ ID NO: 8) PEPPPELENNKTMNRAENGG-C (SEQ ID NO: 9)KTMNRAENGGRPPHHPFETK-C (SEQ ID NO: 10) RPPHHPFETKDVSEYS-C(SEQ ID NO: 11)

Rat SOST:

QGWQAFKNDATEIIPGLREYPEPP (SEQ ID NO: 12) PEPPQELENNQTMNRAENGG(SEQ ID NO: 13) ENGGRPPHHPYDTKDVSEYS (SEQ ID NO: 14)TEIIPGLREYPEPPQELENN (SEQ ID NO: 15)

Rat SOST Peptides with Additional Cys:

QGWQAFKNDATEIIPGLREYPEPP-C (SEQ ID NO: 16) PEPPQELENNQTMNRAENGG-C(SEQ ID NO: 17) ENGGRPPHHPYDTKDVSEYS-C (SEQ ID NO: 18)TEIIPGLREYPEPPQELENN-C (SEQ ID NO: 19)

The following peptides were designed to contain the amino acid portionof core region that was predicted to make contact with BMP proteins.Cysteine was added at the C-terminal end of each peptide for conjugationto KLH, and the conjugated peptides were used for immunization. In theDocking Core N-terminal Peptide an internal cysteine was changed to aserine to avoid double conjugation to KLH.

For Human SOST:

Amino acid sequence without Cys residues added:

Docking_Core_N-terminal_Peptide: (SEQ ID NO: 21) IPDRYRAQRVQLLCPGGEAPDocking_Core_Cterm_Peptide: (SEQ ID NO: 22) QLLCPGGEAPRARKVRLVASDocking_Core_N-terminal_Peptide: (SEQ ID NO: 23) IPDRYRAQRVQLLCPGGEAP-CDocking_Core_Cterm_Peptide: (SEQ ID NO: 24) QLLCPGGEAPRARKVRLVAS-C

For Rat SOST:

Amino acid sequence without Cys residues added or substituted:

Docking_Core_N-terminal_Peptide: (SEQ ID NO: 25) IPDRYRAQRVQLLCPGGDocking_Core_Cterm_Peptide: (SEQ ID NO: 26) PGGAAPRSRKVRLVASPeptide immunogens with Cys added and substituted:Docking_Core_N-terminal_Peptide: (SEQ ID NO: 27) IPDRYRAQRVQLLSPGG-CDocking_Core_Cterm_Peptide: (SEQ ID NO: 28) PGGAAPRSRKVRLVAS-C

Two regions within SOST that potentially interact to form SOSThomodimers include the amino acids with the SOST core region that arenot present in Noggin. Human SOST peptides designed to contain thissequence had a C-terminal or N-terminal Cys that was conjugated to KLH.For the rat SOST peptide, a cysteine was added to the carboxy terminusof the sequence (SEQ ID NO:31). The KLH conjugated peptides were usedfor immunization.

For Human SOST:

CGPARLLPNAIGRGKWWRPS (SEQ ID NO: 29) IGRGKWWRPSGPDFRC (SEQ ID NO: 30)

For Rat SOST:

PNAIGRVKWWRPNGPDFR (SEQ ID NO: 31) Rat SOST peptide with cysteine addedPNAIGRVKWWRPNGPDFR-C (SEQ ID NO: 32)

The second region within SOST that potentially interacts to form SOSThomodimers includes the C-terminal region. Peptide immunogens weredesigned to include amino acid sequences within this region (see below).For conjugation to KLH, a cysteine residue was added to the C-terminalend, and the conjugated peptides were used for immunization.

For Human SOST:

KRLTRFHNQS ELKDFGTEAA (SEQ ID NO: 33) ELKDFGTEAA RPQKGRKPRP(SEQ ID NO: 34) RPQKGRKPRP RARSAKANQA (SEQ ID NO: 35) RARSAKANQA ELENAY(SEQ ID NO: 36)

Peptide Immunogens with Cys Added at C-terminus:

KRLTRFHNQS ELKDFGTEAA-C (SEQ ID NO: 37) ELKDFGTEAA RPQKGRKPRP-C(SEQ ID NO: 38) RPQKGRKPRP RARSAKANQA-C (SEQ ID NO: 39)RARSAKANQA ELENAY-C (SEQ ID NO: 40)

For Rat SOST:

KRLTRFHNQSELKDFGPETARPQ (SEQ ID NO: 41) KGRKPRPRARGAKANQAELENAY(SEQ ID NO: 42) SELKDFGPETARPQKGRKPRPRAR (SEQ ID NO: 43)

Peptide Immunogens with Cys Added at C-terminus:

KRLTRFHNQSELKDFGPETARPQ-C (SEQ ID NO: 44) KGRKPRPRARGAKANQAELENAY-C(SEQ ID NO: 45) SELKDFGPETARPQKGRKPRPRAR-C (SEQ ID NO: 46)

Example 4 Assay for Detecting Binding of Antibodies to a TGF-BetaBinding-Protein

This example describes an assay for detecting binding of a ligand, forexample, an antibody or antibody fragment thereof, to sclerostin.

A FLAG®-sclerostin fusion protein was prepared according to protocolsprovided by the manufacturer (Sigma Aldrich, St. Louis, Mo.) and asdescribed in U.S. Pat. No. 6,395,511. Each well of a 96 well microtiterplate is coated with anti-FLAG® monoclonal antibody (Sigma Aldrich) andthen blocked with 10% BSA in PBS. The fusion protein (20 ng) is added to100 μl PBS/0.2% BSA and adsorbed onto the 96-well plate for 60 minutesat room temperature. This protein solution is removed and the wells arewashed to remove unbound fusion protein. A BMP, for example, BMP-4,BMP-5, BMP-6, or BMP-7, is diluted in PBS/0.2% BSA and added to eachwell at concentrations ranging from 10 μM to 500 nM. After an incubationfor 2 hours at room temperature, the binding solution is removed and theplate is washed three times with 200 μl volumes of PBS/0.2% BSA. Bindingof the BMP to sclerostin is detected using polyclonal antiserum ormonoclonal antibody specific for the BMP and an appropriateenzyme-conjugated second step reagent according to standard ELISAtechniques (see, e.g., Ausubel et al., Current Protocols in Mol. Biol.Vol 2 11.2.1-11.2.22 (1998)). Specific binding is calculated bysubtracting non-specific binding from total binding and analyzed usingthe LIGAND program (Munson and Podbard, Anal. Biochem. 107:220-39(1980)).

Binding of sclerostin to a BMP is also detected by homogeneous timeresolved fluorescence detection (Mellor et al., J. Biomol. Screening,3:91-99 (1998)). A polynucleotide sequence encoding sclerostin isoperatively linked to a human immunoglobulin constant region in arecombinant nucleic acid construct and expressed as a humanFc-sclerostin fusion protein according to methods known in the art anddescribed herein. Similarly, a BMP ligand is engineered and expressed asa BMP-mouse Fc fusion protein. These two fusion proteins are incubatedtogether and the assay conducted as described by Mellor et al.

Example 5 Screening Assay for Antibodies that Inhibit Binding ofTGF-Beta Family Members to TGF-Beta Binding Protein

This example describes a method for detecting an antibody that inhibitsbinding of a TGF-beta family member to sclerostin. An ELISA is performedessentially as described in Example 4 except that the BMP concentrationis held fixed at its Kd (determined, for example, by BIAcore analysis).In addition, an antibody or a library or collection of antibodies isadded to the wells to a concentration of 1 μM. Antibodies are incubatedfor 2 hours at room temperature with the BMP and sclerostin, thesolution removed, and the bound BMP is quantified as described (seeExample 4). Antibodies that inhibit 40% of the BMP binding observed inthe absence of antibody are considered antagonists of this interaction.These antibodies are further evaluated as potential inhibitors byperforming titration studies to determine their inhibition constants andtheir effect on TGF-beta binding-protein binding affinity. Comparablespecificity control assays may also be conducted to establish theselectivity profile for the identified antagonist using assays dependenton the BMP ligand action (e.g., a BMP/BMP receptor competition study).

Example 6 Inhibition of TGF-Beta Binding-Protein Localization to BoneMatrix

Evaluation of inhibition of localization to bone matrix (hydroxyapatite)is conducted using modifications to the method of Nicolas (Calcif TissueInt. 57:206-12 (1995)). Briefly, ¹²⁵I-labelled TGF-beta binding-proteinis prepared as described by Nicolas (supra). Hydroxyapatite is added toeach well of a 96-well microtiter plate equipped with a polypropylenefiltration membrane (Polyfiltroninc, Weymouth Mass.). TGF-betabinding-protein diluted in 0.2% albumin in PBS buffer is then added tothe wells. The wells containing matrix are washed 3 times with 0.2%albumin in PBS buffer. Adsorbed TGF-beta binding-protein is eluted using0.3 M NaOH and then quantified.

An antibody or other agent that inhibits or impairs binding of thesclerostin TGF-beta binding protein to the hydroxyapatite is identifiedby incubating the TGF-beta binding protein with the antibody andapplying the mixture to the matrix as described above. The matrix iswashed 3 times with 0.2% albumin in PBS buffer. Adsorbed sclerostin iseluted with 0.3 M NaOH and then quantified. An antibody that inhibitsthe level of binding of sclerostin to the hydroxyapatite by at least 40%compared to the level of binding observed in the absence of antibody isconsidered a bone localization inhibitor. Such an antibody is furthercharacterized in dose response studies to determine its inhibitionconstant and its effect on TGF-beta binding-protein binding affinity.

From the foregoing, although specific embodiments of the invention havebeen described herein for purposes of illustration, variousmodifications may be made without deviating from the spirit and scope ofthe invention. Accordingly, the invention is not limited except as bythe appended claims.

1.-51. (canceled)
 52. An isolated antibody, or antigen-binding fragmentthereof, that binds to a sclerostin polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 1, wherein said antibody or fragmentbinds to the sequence of SEQ ID NO:
 53. 53. The antibody orantigen-binding fragment of claim 52 that is capable of increasing bonemineral content in a mammal.
 54. The antibody or antigen-bindingfragment of claim 52, wherein the antibody is a monoclonal antibody. 55.The antibody or antigen-binding fragment of claim 52, wherein theantibody is a human antibody, a humanized antibody or a chimericantibody.
 56. The antibody or antigen-binding fragment of claim 52 thatcomprises an antigen-binding fragment.
 57. The antibody orantigen-binding fragment of claim 56, wherein the antigen-bindingfragment is selected from the group consisting of F(ab′)2, Fab, Fab′,Fd, and Fv.
 58. A hybridoma cell producing the antibody orantigen-binding fragment of claim
 52. 59. A nucleic acid comprising anucleic acid sequence encoding the antibody or antigen-binding fragmentof claim
 52. 60. An isolated host cell comprising the nucleic acid ofclaim
 59. 61. A vector comprising the nucleic acid of claim
 59. 62. Anisolated host cell comprising the vector of claim
 61. 63. A method ofproducing an antibody or antigen-binding fragment thereof comprising thestep of culturing the host cell of claim 60 and isolating the antibodyproduced therefrom.
 64. A method of producing an antibody orantigen-binding fragment thereof comprising the step of culturing thehost cell of claim 62 and isolating the antibody produced therefrom. 65.A composition comprising the antibody or antigen-binding fragment ofclaim 52 and a pharmaceutically acceptable carrier.
 66. A method forincreasing bone mineral content in a warm-blooded animal, the methodcomprising administering to the warm-blooded animal the antibody orantigen-binding fragment of claim
 52. 67. The method of claim 66,wherein the warm-blooded animal is a human.
 68. The method of claim 67,wherein the human has a condition selected from the group consisting ofachondroplasia, cleidrocranial dysostosise, echondromatosis, fibrousdysplasia, Gaucher's hypophosphatemic rickets, Marfan's, multiplehereditary exotoses, neurofibromatosis, osteogenesis inperfecta,osteopetrosis, osteopoikilosis sclerotic lesions, fractures, periodontaldisease, pseudoarthosis and pyogenic osteolmyelitis.
 69. A method oftreating bone fracture, dysplasia associated with abnormal growth ordevelopment of bone, osteoporosis, or osteopenia in a human wherein themethod comprises administering to the human the antibody orantigen-binding fragment of claim
 52. 70. The method of claim 69,wherein the osteopenia is caused by an anaemic state, steroids, heparin,a bone morrow disorder, scurvy, malnutrition, calcium deficiency,idiopathic osteoporosis, congenital osteopenia or osteoporosis,alcoholism, chronic liver disease, senility, post menstrual state,oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus,hyperthyroidism, Cushing's disease, acromegaly, hypogonadism,immobilization or disuse, reflex sympathetic dystrophy syndrome,transient regional osteoporosis or osteomalacia.
 71. An isolatedantibody, or antigen binding fragment thereof, that binds to the aminoacid sequence of SEQ ID NO: 53 within SEQ ID NO: 1 with an affinityK_(D) of less than or equal to about 10⁻⁶ M.
 72. The antibody orantigen-binding fragment of claim 71 that binds to a second amino acidsequence of SEQ ID NO: 1, wherein said second amino acid sequence is SEQID NO: 22 or SEQ ID NO:
 33. 73. The antibody or antigen-binding fragmentof claim 71 that is capable of increasing bone mineral content in amammal.
 74. The antibody or antigen-binding fragment of claim 71, hereinthe antibody is a monoclonal antibody.
 75. The antibody orantigen-binding fragment of claim 71, wherein the antibody is a humanantibody, a humanized antibody or a chimeric antibody.
 76. The antibodyor antigen-binding fragment of claim 71 that comprises anantigen-binding fragment.
 77. The antibody or antigen-binding fragmentof claim 76, wherein the antigen-binding fragment is selected from thegroup consisting of F(ab′)₂, Fab, Fab′, Fd, and Fv.
 78. A hybridoma cellproducing the antibody or antigen-binding fragment of claim
 71. 79. Anucleic acid comprising a nucleic acid sequence encoding the antibody orantigen-binding fragment of claim
 71. 80. An isolated host cellcomprising the nucleic acid of claim
 79. 81. A vector comprising thenucleic acid of claim
 79. 82. An isolated host cell comprising thevector of claim
 81. 83. A method of producing an antibody comprising thestep of culturing the host cell of claim 80 and isolating the antibodyproduced therefrom.
 84. A method of producing an antibody comprising thestep of culturing the host cell of claim 82 and isolating the antibodyproduced therefrom.
 85. A composition comprising the antibody orantigen-binding fragment of claim 71 and a pharmaceutically acceptablecarrier.
 86. A method for increasing bone mineral content in awarm-blooded animal, the method comprising administering to thewarm-blooded animal the antibody or antigen-binding fragment of claim71.
 87. The method of claim 86, wherein the warm-blooded animal is ahuman.
 88. The method of claim 87, wherein the human has a conditionselected from the group consisting of achondroplasia, cleidrocranialdysostosise, echondromatosis, fibrous dysplasia, Gaucher'shypophosphatemic rickets, Marfan's, multiple hereditary exotoses,neurofibromatosis, osteogenesis inperfecta, osteopetrosis,osteopoikilosis sclerotic lesions, fractures, periodontal disease,pseudoarthosis and pyogenic osteomyelitis.
 89. A method of treating bonefracture, dysplasia associated with abnormal growth or development ofbone, osteoporosis, or osteopenia in a human wherein the methodcomprises administering to the human the antibody or antigen-bindingfragment of claim
 71. 90. The method of claim 89, wherein the osteopeniais caused by an anaemic state, steroids, heparin, a bone morrowdisorder, scurvy, malnutrition, calcium deficiency, idiopathicosteoporosis, congenital osteopenia or osteoporosis, alcoholism, chronicliver disease, senility, post menstrual state, oligomenorrhea,amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, Cushing'sdisease, acromegaly, hypogonadism, immobilization or disuse, reflexsympathetic dystrophy syndrome, transient regional osteoporosis orosteomalacia.